Publications by authors named "Andrew A Walker"

30 Publications

  • Page 1 of 1

Venom chemistry underlying the painful stings of velvet ants (Hymenoptera: Mutillidae).

Cell Mol Life Sci 2021 May 10. Epub 2021 May 10.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, 4072, Australia.

Velvet ants (Hymenoptera: Mutillidae) are a family of solitary parasitoid wasps that are renowned for their painful stings. We explored the chemistry underlying the stings of mutillid wasps of the genus Dasymutilla Ashmead. Detailed analyses of the venom composition of five species revealed that they are composed primarily of peptides. We found that two kinds of mutillid venom peptide appear to be primarily responsible for the painful effects of envenomation. These same peptides also have defensive utility against invertebrates, since they were able to incapacitate and kill honeybees. Both act directly on cell membranes where they directly increase ion conductivity. The defensive venom peptides of Dasymutilla bear a striking similarity, in structure and mode of action, to those of the ant Myrmecia gulosa (Fabricius), suggesting either retention of ancestral toxins, or convergence driven by similar life histories and defensive selection pressures. Finally, we propose that other highly expressed Dasymutilla venom peptides may play a role in parasitisation, possible in delay or arrest of host development. This study represents the first detailed account of the composition and function of the venoms of the Mutillidae.
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http://dx.doi.org/10.1007/s00018-021-03847-1DOI Listing
May 2021

Production, composition, and mode of action of the painful defensive venom produced by a limacodid caterpillar, .

Proc Natl Acad Sci U S A 2021 May;118(18)

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia;

Venoms have evolved independently several times in Lepidoptera. Limacodidae is a family with worldwide distribution, many of which are venomous in the larval stage, but the composition and mode of action of their venom is unknown. Here, we use imaging technologies, transcriptomics, proteomics, and functional assays to provide a holistic picture of the venom system of a limacodid caterpillar, Contrary to dogma that defensive venoms are simple in composition, produces a complex venom containing 151 proteinaceous toxins spanning 59 families, most of which are peptides <10 kDa. Three of the most abundant families of venom peptides (vulnericins) are 1) analogs of the adipokinetic hormone/corazonin-related neuropeptide, some of which are picomolar agonists of the endogenous insect receptor; 2) linear cationic peptides derived from cecropin, an insect innate immune peptide that kills bacteria and parasites by disrupting cell membranes; and 3) disulfide-rich knottins similar to those that dominate spider venoms. Using venom fractionation and a suite of synthetic venom peptides, we demonstrate that the cecropin-like peptides are responsible for the dominant pain effect observed in mammalian in vitro and in vivo nociception assays and therefore are likely to cause pain after natural envenomations by Our data reveal convergent molecular evolution between limacodids, hymenopterans, and arachnids and demonstrate that lepidopteran venoms are an untapped source of novel bioactive peptides.
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http://dx.doi.org/10.1073/pnas.2023815118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8106304PMC
May 2021

Crouching Tiger, Hidden Protein: Searching for Insecticidal Toxins in Venom of the Red Tiger Assassin Bug ().

Toxins (Basel) 2020 Dec 22;13(1). Epub 2020 Dec 22.

Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Rd, St Lucia, QLD 4072, Australia.

Assassin bugs are venomous insects that prey on other arthropods. Their venom has lethal, paralytic, and liquifying effects when injected into prey, but the toxins responsible for these effects are unknown. To identify bioactive assassin bug toxins, venom was harvested from the red tiger assassin bug (), an Australian species whose venom has not previously been characterised. The venom was fractionated using reversed-phase high-performance liquid chromatography, and four fractions were found to cause paralysis and death when injected into sheep blowflies (). The amino acid sequences of the major proteins in two of these fractions were elucidated by comparing liquid chromatography/tandem mass spectrometry data with a translated venom-gland transcriptome. The most abundant components were identified as a solitary 12.8 kDa CUB (complement C1r/C1s, Uegf, Bmp1) domain protein and a 9.5 kDa cystatin. CUB domains are present in multidomain proteins with diverse functions, including insect proteases. Although solitary CUB domain proteins have been reported to exist in other heteropteran venoms, such as that of the bee killer assassin bug , their function is unknown, and they have not previously been reported as lethal or paralysis-inducing. Cystatins occur in the venoms of spiders and snakes, but again with an unknown function. Reduction and alkylation experiments revealed that the venom cystatin featured five cysteine residues, one of which featured a free sulfhydryl group. These data suggest that solitary CUB domain proteins and/or cystatins may contribute to the insecticidal activity of assassin bug venom.
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http://dx.doi.org/10.3390/toxins13010003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822193PMC
December 2020

Two for the Price of One: Heterobivalent Ligand Design Targeting Two Binding Sites on Voltage-Gated Sodium Channels Slows Ligand Dissociation and Enhances Potency.

J Med Chem 2020 11 20;63(21):12773-12785. Epub 2020 Oct 20.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD 4072, Australia.

Voltage-gated sodium (Na) channels are pore-forming transmembrane proteins that play essential roles in excitable cells, and they are key targets for antiepileptic, antiarrhythmic, and analgesic drugs. We implemented a heterobivalent design strategy to modulate the potency, selectivity, and binding kinetics of Na channel ligands. We conjugated μ-conotoxin KIIIA, which occludes the pore of the Na channels, to an analogue of huwentoxin-IV, a spider-venom peptide that allosterically modulates channel gating. Bioorthogonal hydrazide and copper-assisted azide-alkyne cycloaddition conjugation chemistries were employed to generate heterobivalent ligands using polyethylene glycol linkers spanning 40-120 Å. The ligand with an 80 Å linker had the most pronounced bivalent effects, with a significantly slower dissociation rate and 4-24-fold higher potency compared to those of the monovalent peptides for the human Na1.4 channel. This study highlights the power of heterobivalent ligand design and expands the repertoire of pharmacological probes for exploring the function of Na channels.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667638PMC
November 2020

Deadly Proteomes: A Practical Guide to Proteotranscriptomics of Animal Venoms.

Proteomics 2020 09;20(17-18):e1900324

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland, 4072, Australia.

Animal venoms are renowned for their toxicity, biochemical complexity, and as a source of compounds with potential applications in medicine, agriculture, and industry. Polypeptides underlie much of the pharmacology of animal venoms, and elucidating these arsenals of polypeptide toxins-known as the venom proteome or venome-is an important step in venom research. Proteomics is used for the identification of venom toxins, determination of their primary structure including post-translational modifications, as well as investigations into the physiology underlying their production and delivery. Advances in proteomics and adjacent technologies has led to a recent upsurge in publications reporting venom proteomes. Improved mass spectrometers, better proteomic workflows, and the integration of next-generation sequencing of venom-gland transcriptomes and venomous animal genomes allow quicker and more accurate profiling of venom proteomes with greatly reduced starting material. Technologies such as imaging mass spectrometry are revealing additional insights into the mechanism, location, and kinetics of venom toxin production. However, these numerous new developments may be overwhelming for researchers designing venom proteome studies. Here, the field of venom proteomics is reviewed and some practical solutions for simplifying mass spectrometry workflows to study animal venoms are offered.
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http://dx.doi.org/10.1002/pmic.201900324DOI Listing
September 2020

The evolutionary dynamics of venom toxins made by insects and other animals.

Authors:
Andrew A Walker

Biochem Soc Trans 2020 08;48(4):1353-1365

Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia.

Animal venoms are recognised as unique biological systems in which to study molecular evolution. Venom use has evolved numerous times among the insects, and insects today use venom to capture prey, defend themselves from predators, or to subdue and modulate host responses during parasitism. However, little is known about most insect venom toxins or the mode and tempo by which they evolve. Here, I review the evolutionary dynamics of insect venom toxins, and argue that insects offer many opportunities to examine novel aspects of toxin evolution. The key questions addressed are: How do venomous animals evolve from non-venomous animals, and how does this path effect the composition and pharmacology of the venom? What genetic processes (gene duplication, co-option, neofunctionalisation) are most important in toxin evolution? What kinds of selection pressures are acting on toxin-encoding genes and their cognate targets in envenomated animals? The emerging evidence highlights that venom composition and pharmacology adapts quickly in response to changing selection pressures resulting from new ecological interactions, and that such evolution occurs through a stunning variety of genetic mechanisms. Insects offer many opportunities to investigate the evolutionary dynamics of venom toxins due to their evolutionary history rich in venom-related adaptations, and their quick generation time and suitability for culture in the laboratory.
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http://dx.doi.org/10.1042/BST20190820DOI Listing
August 2020

Venom Peptides with Dual Modulatory Activity on the Voltage-Gated Sodium Channel Na1.1 Provide Novel Leads for Development of Antiepileptic Drugs.

ACS Pharmacol Transl Sci 2020 Feb 25;3(1):119-134. Epub 2019 Nov 25.

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland 4072, Australia.

Voltage-gated sodium (Na) channels play a fundamental role in normal neurological function, especially via the initiation and propagation of action potentials. The Na1.1 subtype is found in inhibitory interneurons of the brain and it is essential for maintaining a balance between excitation and inhibition in neuronal networks. Heterozygous loss-of-function mutations of , the gene encoding Na1.1, underlie Dravet syndrome (DS), a severe pediatric epilepsy. We recently demonstrated that selective inhibition of Na1.1 inactivation prevents seizures and premature death in a mouse model of DS. Thus, selective modulators of Na1.1 might be useful therapeutics for treatment of DS as they target the underlying molecular deficit. Numerous scorpion-venom peptides have been shown to modulate the activity of Na channels, but little is known about their activity at Na1.1. Here we report the isolation, sequence, three-dimensional structure, recombinant production, and functional characterization of two peptidic modulators of Na1.1 from venom of the buthid scorpion . These peptides, Hj1a and Hj2a, are potent agonists of Na1.1 (EC of 17 and 32 nM, respectively), and they present dual α/β activity by modifying both the activation and inactivation properties of the channel. NMR studies of rHj1a indicate that it adopts a cystine-stabilized αβ fold similar to known scorpion toxins. Although Hj1a and Hj2a have only limited selectivity for Na1.1, their unusual dual mode of action provides an alternative approach to the development of selective Na1.1 modulators for the treatment of DS.
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http://dx.doi.org/10.1021/acsptsci.9b00079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088997PMC
February 2020

Weaponisation 'on the fly': Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies.

Insect Biochem Mol Biol 2020 03 21;118:103310. Epub 2019 Dec 21.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia. Electronic address:

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the K11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of K11.1 channel activation to more depolarised potentials, but has no effect on K1.3, K2.1, K10.1, K1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these K channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.
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http://dx.doi.org/10.1016/j.ibmb.2019.103310DOI Listing
March 2020

Missiles of Mass Disruption: Composition and Glandular Origin of Venom Used as a Projectile Defensive Weapon by the Assassin Bug .

Toxins (Basel) 2019 11 18;11(11). Epub 2019 Nov 18.

Institute for Molecular Bioscience, the University of Queensland, St. Lucia, Queensland 4072, Australia.

Assassin bugs (Reduviidae) produce venoms that are insecticidal, and which induce pain in predators, but the composition and function of their individual venom components is poorly understood. We report findings on the venom system of the red-spotted assassin bug , a large species of African origin that is unique in propelling venom as a projectile weapon when threatened. We performed RNA sequencing experiments on venom glands (separate transcriptomes of the posterior main gland, PMG, and the anterior main gland, AMG), and proteomic experiments on venom that was either defensively propelled or collected from the proboscis in response to electrostimulation. We resolved a venom proteome comprising 166 polypeptides. Both defensively propelled venom and most venom samples collected in response to electrostimulation show a protein profile similar to the predicted secretory products of the PMG, with a smaller contribution from the AMG. Pooled venom samples induce calcium influx via membrane lysis when applied to mammalian neuronal cells, consistent with their ability to cause pain when propelled into the eyes or mucus membranes of potential predators. The same venom induces rapid paralysis and death when injected into fruit flies. These data suggest that the cytolytic, insecticidal venom used by reduviids to capture prey is also a highly effective defensive weapon when propelled at predators.
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http://dx.doi.org/10.3390/toxins11110673DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6891600PMC
November 2019

Buzz Kill: Function and Proteomic Composition of Venom from the Giant Assassin Fly (Diptera: Asilidae).

Toxins (Basel) 2018 Nov 5;10(11). Epub 2018 Nov 5.

Venom Evolution Lab, School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia.

Assassin flies (Diptera: Asilidae) inject paralysing venom into insect prey during hunting, but their venoms are poorly characterised in comparison to those produced by spiders, scorpions, or hymenopteran insects. Here we investigated the composition of the venom of the giant Australian assassin fly using a combination of insect microinjection assays, calcium imaging assays of mammalian sensory neurons, proteomics and transcriptomics. Injection of venom into blowflies () produced rapid contractile paralysis (PD at 1 min = 3.1 μg per fly) followed by death, and also caused immediate activation of mouse dorsal root ganglion neurons (at 50 ng/μL). These results are consistent with venom use for both prey capture and predator deterrence. Paragon searches of tandem mass spectra of venom against a translated thoracic gland RNA-Seq database identified 122 polypeptides present in the venom, including six linear and 21 disulfide-rich peptides. Some of these disulfide-rich peptides display sequence homology to peptide families independently recruited into other animal venoms, including inhibitor cystine knots, cystine-stabilised α/β defensins, Kazal peptides, and von Willebrand factors. Numerous enzymes are present in the venom, including 35 proteases of the S1 family, proteases of the S10, C1A, M12A, M14, and M17 families, and phosphatase, amylase, hydrolase, nuclease, and dehydrogenase-like proteins. These results highlight convergent molecular evolution between the assassin flies and other venomous animals, as well as the unique and rich molecular composition of assassin fly venom.
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http://dx.doi.org/10.3390/toxins10110456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266666PMC
November 2018

Entomo-venomics: The evolution, biology and biochemistry of insect venoms.

Toxicon 2018 Nov 26;154:15-27. Epub 2018 Sep 26.

Institute for Molecular Bioscience, The University of Queensland, St. Lucia, QLD 4072, Australia. Electronic address:

The insects are a hyperdiverse class containing more species than all other animal groups combined-and many employ venom to capture prey, deter predators and micro-organisms, or facilitate parasitism or extra-oral digestion. However, with the exception of those made by Hymenoptera (wasps, ants and bees), little is known about insect venoms. Here, we review the current literature on insects that use venom for prey capture and predator deterrence, finding evidence for fourteen independent origins of venom usage among insects, mostly among the hyperdiverse holometabolan orders. Many lineages, including the True Bugs (Heteroptera), robber flies (Asilidae), and larvae of many Neuroptera, Coleoptera and Diptera, use mouthpart-associated venoms to paralyse and pre-digest prey during hunting. In contrast, some Hymenoptera and larval Lepidoptera, and one species of beetle, use non-mouthpart structures to inject venom in order to cause pain to deter potential predators. Several recently published insect venom proteomes indicate molecular convergence between insects and other venomous animal groups, with all insect venoms studied so far being potently bioactive cocktails containing both peptides and larger proteins, including novel peptide and protein families. This review summarises the current state of the field of entomo-venomics.
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http://dx.doi.org/10.1016/j.toxicon.2018.09.004DOI Listing
November 2018

The assassin bug Pristhesancus plagipennis produces two distinct venoms in separate gland lumens.

Nat Commun 2018 02 22;9(1):755. Epub 2018 Feb 22.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia.

The assassin bug venom system plays diverse roles in prey capture, defence and extra-oral digestion, but it is poorly characterised, partly due to its anatomical complexity. Here we demonstrate that this complexity results from numerous adaptations that enable assassin bugs to modulate the composition of their venom in a context-dependent manner. Gland reconstructions from multimodal imaging reveal three distinct venom gland lumens: the anterior main gland (AMG); posterior main gland (PMG); and accessory gland (AG). Transcriptomic and proteomic experiments demonstrate that the AMG and PMG produce and accumulate distinct sets of venom proteins and peptides. PMG venom, which can be elicited by electrostimulation, potently paralyses and kills prey insects. In contrast, AMG venom elicited by harassment does not paralyse prey insects, suggesting a defensive role. Our data suggest that assassin bugs produce offensive and defensive venoms in anatomically distinct glands, an evolutionary adaptation that, to our knowledge, has not been described for any other venomous animal.
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http://dx.doi.org/10.1038/s41467-018-03091-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823883PMC
February 2018

Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.

Cell Mol Life Sci 2018 09 9;75(17):3215-3229. Epub 2018 Feb 9.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia.

True Bugs (Insecta: Heteroptera) produce venom or saliva with diverse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins underlying these biological activities. We examined venom of the giant fish-killing water bug Lethocerus distinctifemur (Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and diversified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.
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http://dx.doi.org/10.1007/s00018-018-2768-1DOI Listing
September 2018

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug (Hemiptera: Reduviidae).

Mol Cell Proteomics 2017 04 27;16(4):552-566. Epub 2017 Jan 27.

From the ‡Institute for Molecular Bioscience,

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.
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http://dx.doi.org/10.1074/mcp.M116.063321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5383778PMC
April 2017

Venoms of Heteropteran Insects: A Treasure Trove of Diverse Pharmacological Toolkits.

Toxins (Basel) 2016 Feb 12;8(2):43. Epub 2016 Feb 12.

Institute for Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, Australia.

The piercing-sucking mouthparts of the true bugs (Insecta: Hemiptera: Heteroptera) have allowed diversification from a plant-feeding ancestor into a wide range of trophic strategies that include predation and blood-feeding. Crucial to the success of each of these strategies is the injection of venom. Here we review the current state of knowledge with regard to heteropteran venoms. Predaceous species produce venoms that induce rapid paralysis and liquefaction. These venoms are powerfully insecticidal, and may cause paralysis or death when injected into vertebrates. Disulfide-rich peptides, bioactive phospholipids, small molecules such as N,N-dimethylaniline and 1,2,5-trithiepane, and toxic enzymes such as phospholipase A2, have been reported in predatory venoms. However, the detailed composition and molecular targets of predatory venoms are largely unknown. In contrast, recent research into blood-feeding heteropterans has revealed the structure and function of many protein and non-protein components that facilitate acquisition of blood meals. Blood-feeding venoms lack paralytic or liquefying activity but instead are cocktails of pharmacological modulators that disable the host haemostatic systems simultaneously at multiple points. The multiple ways venom is used by heteropterans suggests that further study will reveal heteropteran venom components with a wide range of bioactivities that may be recruited for use as bioinsecticides, human therapeutics, and pharmacological tools.
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http://dx.doi.org/10.3390/toxins8020043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4773796PMC
February 2016

More than one way to spin a crystallite: multiple trajectories through liquid crystallinity to solid silk.

Proc Biol Sci 2015 Jun;282(1809):20150259

Food and Nutrition, CSIRO, Canberra 2600, Australia

Arthropods face several key challenges in processing concentrated feedstocks of proteins (silk dope) into solid, semi-crystalline silk fibres. Strikingly, independently evolved lineages of silk-producing organisms have converged on the use of liquid crystal intermediates (mesophases) to reduce the viscosity of silk dope and assist the formation of supramolecular structure. However, the exact nature of the liquid-crystal-forming-units (mesogens) in silk dope, and the relationship between liquid crystallinity, protein structure and silk processing is yet to be fully elucidated. In this review, we focus on emerging differences in this area between the canonical silks containing extended-β-sheets made by silkworms and spiders, and 'non-canonical' silks made by other insect taxa in which the final crystallites are coiled-coils, collagen helices or cross-β-sheets. We compared the amino acid sequences and processing of natural, regenerated and recombinant silk proteins, finding that canonical and non-canonical silk proteins show marked differences in length, architecture, amino acid content and protein folding. Canonical silk proteins are long, flexible in solution and amphipathic; these features allow them both to form large, micelle-like mesogens in solution, and to transition to a crystallite-containing form due to mechanical deformation near the liquid-solid transition. By contrast, non-canonical silk proteins are short and have rod or lath-like structures that are well suited to act both as mesogens and as crystallites without a major intervening phase transition. Given many non-canonical silk proteins can be produced at high yield in E. coli, and that mesophase formation is a versatile way to direct numerous kinds of supramolecular structure, further elucidation of the natural processing of non-canonical silk proteins may to lead to new developments in the production of advanced protein materials.
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http://dx.doi.org/10.1098/rspb.2015.0259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4590440PMC
June 2015

The other prey-capture silk: Fibres made by glow-worms (Diptera: Keroplatidae) comprise cross-β-sheet crystallites in an abundant amorphous fraction.

Comp Biochem Physiol B Biochem Mol Biol 2015 Sep 22;187:78-84. Epub 2015 May 22.

Ecosystem Sciences, Commonwealth Scientific and Industrial Research Organisation, Black Mountain Laboratories, Canberra, 2601, Australia. Electronic address:

Glow-worms (larvae of dipteran genus Arachnocampa) are restricted to moist habitats where they capture flying prey using snares composed of highly extensible silk fibres and sticky mucus droplets. Little is known about the composition or structure of glow-worm snares, or the extent of possible convergence between glow-worm and arachnid capture silks. We characterised Arachnocampa richardsae silk and mucus using X-ray scattering, Fourier transform infrared spectroscopy and amino acid analysis. Silk but not mucus contained crystallites of the cross-β-sheet type, which occur in unrelated insect silks but have not been reported previously in fibres used for prey capture. Mucus proteins were rich in Gly (28.5%) and existed in predominantly a random coil structure, typical of many adhesive proteins. In contrast, the silk fibres were unusually rich in charged and polar residues, particularly Lys (18.1%), which we propose is related to their use in a highly hydrated state. Comparison of X-ray scattering, infrared spectroscopy and amino acid analysis data suggests that silk fibres contain a high fraction of disordered protein. We suggest that in the native hydrated state, silk fibres are capable of extension via deformation of both disordered regions and cross-β-sheet crystallites, and that high extensibility is an adaptation promoting successful prey capture. This study illustrates the rich variety of protein motifs that are available for recruitment into biopolymers, and how convergently evolved materials can nevertheless be based on fundamentally different protein structures.
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http://dx.doi.org/10.1016/j.cbpb.2015.05.008DOI Listing
September 2015

Convergently-evolved structural anomalies in the coiled coil domains of insect silk proteins.

J Struct Biol 2014 Jun 13;186(3):402-11. Epub 2014 Jan 13.

Materials Science and Engineering, CSIRO, Australia.

The use of coiled coil proteins as the basis of silk materials is an engineering solution that has evolved convergently in at least five insect lineages-the stinging hymenopterans (ants, bees, hornets), argid sawflies, fleas, lacewings, and praying mantises-and persisted throughout large radiations of these insect families. These coiled coil silk proteins share a characteristic distinct from other coiled coil proteins, in that they are fabricated into solid materials after accumulating as highly concentrated solutions within dedicated glands. Here, we relate the amino acid sequences of these proteins to the secondary and tertiary structural information available from biophysical methods such as X-ray scattering, nuclear magnetic resonance and Raman spectroscopy. We investigate conserved and convergently evolved features within these proteins and compare these to the features of classic coiled coil proteins including tropomyosin and leucine zippers. Our analysis finds that the coiled coil domains of insect silk proteins have several common structural anomalies including a high prevalence of alanine residues in core positions. These atypical features of the coiled coil fibrous proteins - which likely produce deviations from canonical coiled-coil structure - likely exist due to selection pressures related to the process of silk fabrication and the final function of the proteins.
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http://dx.doi.org/10.1016/j.jsb.2014.01.002DOI Listing
June 2014

A comparison of convergently evolved insect silks that share β-sheet molecular structure.

Biopolymers 2014 Jun;101(6):630-9

CSIRO Materials Science and Engineering, Pigdons Road, Waurn, Ponds, VIC 3216, Australia.

Raspy crickets produce silk webs that are used to build shelters. These webs have been found to consist of both fiber and film components. Raman spectra obtained from both components were found to be very similar for a given species. The protein structure of the fibers and films produced by both species was predominately β-sheet with lesser amounts of β-turns, unordered and α-helical protein also detected. The orientation of the β-sheet backbone in the fiber was determined to be parallel to the fiber axis. Compared to cocoon and dragline silk the orientation distribution exhibits a significant randomly orientated protein component. Amino acid analysis confirmed the presence of glycine, serine, and alanine in both species, which are known to form antiparallel β-sheet structures. Both species, although at significantly different concentrations, where found to contain proline. This amino acid is uncommon in insect silks, and likely involved in increasing fiber elasticity.
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http://dx.doi.org/10.1002/bip.22431DOI Listing
June 2014

A new class of animal collagen masquerading as an insect silk.

Sci Rep 2013 Oct 4;3:2864. Epub 2013 Oct 4.

CSIRO Ecosystem Sciences, Clunies Ross Street, Acton, ACT, 2601, Australia.

Collagen is ubiquitous throughout the animal kingdom, where it comprises some 28 diverse molecules that form the extracellular matrix within organisms. In the 1960s, an extracorporeal animal collagen that forms the cocoon of a small group of hymenopteran insects was postulated. Here we categorically demonstrate that the larvae of a sawfly species produce silk from three small collagen proteins. The native proteins do not contain hydroxyproline, a post translational modification normally considered characteristic of animal collagens. The function of the proteins as silks explains their unusual collagen features. Recombinant proteins could be produced in standard bacterial expression systems and assembled into stable collagen molecules, opening the door to manufacture a new class of artificial collagen materials.
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http://dx.doi.org/10.1038/srep02864DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3790195PMC
October 2013

FAST INdiCATE Trial protocol. Clinical efficacy of functional strength training for upper limb motor recovery early after stroke: neural correlates and prognostic indicators.

Int J Stroke 2014 Feb 12;9(2):240-5. Epub 2013 Sep 12.

Faculty of Medicine and Health Sciences, University of East Anglia, Norwich, UK.

Rationale: Functional strength training in addition to conventional physical therapy could enhance upper limb recovery early after stroke more than movement performance therapy plus conventional physical therapy.

Aims: To determine (a) the relative clinical efficacy of conventional physical therapy combined with functional strength training and conventional physical therapy combined with movement performance therapy for upper limb recovery; (b) the neural correlates of response to conventional physical therapy combined with functional strength training and conventional physical therapy combined with movement performance therapy; (c) whether any one or combination of baseline measures predict motor improvement in response to conventional physical therapy combined with functional strength training or conventional physical therapy combined with movement performance therapy.

Design: Randomized, controlled, observer-blind trial.

Study: The sample will consist of 288 participants with upper limb paresis resulting from a stroke that occurred within the previous 60 days. All will be allocated to conventional physical therapy combined with functional strength training or conventional physical therapy combined with movement performance therapy. Functional strength training and movement performance therapy will be undertaken for up to 1·5 h/day, five-days/week for six-weeks.

Outcomes And Analysis: Measurements will be undertaken before randomization, six-weeks thereafter, and six-months after stroke. Primary efficacy outcome will be the Action Research Arm Test. Explanatory measurements will include voxel-wise estimates of brain activity during hand movement, brain white matter integrity (fractional anisotropy), and brain-muscle connectivity (e.g. latency of motor evoked potentials). The primary clinical efficacy analysis will compare treatment groups using a multilevel normal linear model adjusting for stratification variables and for which therapist administered the treatment. Effect of conventional physical therapy combined with functional strength training versus conventional physical therapy combined with movement performance therapy will be summarized using the adjusted mean difference and 95% confidence interval. To identify the neural correlates of improvement in both groups, we will investigate associations between change from baseline in clinical outcomes and each explanatory measure. To identify baseline measurements that independently predict motor improvement, we will develop a multiple regression model.
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http://dx.doi.org/10.1111/ijs.12179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4228758PMC
February 2014

Micellar refolding of coiled-coil honeybee silk proteins.

J Mater Chem B 2013 Aug 18;1(30):3644-3651. Epub 2013 Jun 18.

CSIRO Ecosystem Sciences, Clunies Ross St, Acton, GPO Box 1700, Canberra, ACT 2601, Australia.

There is growing interest in materials generated from coiled coil proteins for medical applications. In this study we describe controlled micellar refolding of coiled coil honeybee silk proteins using the detergent SDS. Circular dichroism and dynamic light scattering experiments demonstrate that micellar SDS promotes folding of randomly coiled honeybee silk proteins into isolated α-helices, and that removal of detergent micelles, or addition of salt, leads to coiled coil formation. Comparative molecular dynamics simulations of protein helices, with and without SDS, have allowed us to characterize detergent-protein interactions and propose a mechanism of protein folding. In the presence of micellar detergent, hydrophobic residues are associated with the detergent tail groups within the micelles whereas hydrophilic residues are paired with the detergent head-groups on the micelle's surface. These detergent-protein interactions prevent residue-residue interactions and allow the protein to fold according to the natural tendency of individual residues. From this condition, when hydrophobic residue-micellar interactions are reduced by lowering detergent levels to below the critical micelle concentration (CMC) or by using salt to increase detergent packing in micelles and thereby excluding the protein from the interior, the proteins fold into coiled coils. We propose that under low SDS conditions, hydrophobic-monomeric SDS tail-group and hydrophilic-monomeric head-group interactions (low SDS conditions) or hydrophilic-micellar SDS head-group interactions (high salt conditions) stabilize a transient α-helix intermediate in coiled coil folding. The folding pathway constitutes a new kind of micellar refolding, which may be profitably employed to refold other proteins rich in coiled coils.
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http://dx.doi.org/10.1039/c3tb20611dDOI Listing
August 2013

Silverfish silk is formed by entanglement of randomly coiled protein chains.

Insect Biochem Mol Biol 2013 Jul 9;43(7):572-9. Epub 2013 Apr 9.

Research School of Biology, Australian National University, Canberra, ACT 0200, Australia.

Silks are semi-crystalline solids in which protein chains are associated by intermolecular hydrogen bonding within ordered crystallites, and by entanglement within unordered regions. By varying the type of protein secondary structure within crystallites and the overall degree of molecular order within fibers, arthropods produce fibers with a variety of physical properties suited to many purposes. We characterized silk produced as a tactile stimulus during mating by the grey silverfish (Ctenolepisma longicaudata) using Fourier transform infrared spectroscopy, polarized Raman spectroscopy, gel electrophoresis and amino acid analysis. Fibers were proteinaceous-the main component being a 220 kDa protein-and were rich in Gln/Glu, Leu, and Lys. The protein structure present was predominantly random coil, with a lesser amount of beta-structure. Silk fibers could readily be solubilized in aqueous solutions of a mild chaotrope, sodium dodecyl sulfate, indicating protein chains were not cross-linked by disulfide or other covalent bonds. We conclude that entanglement is the major mechanism by which these silk proteins cohere into a solid material. We propose silks used as short-term tactile cues are subject to less stringent requirements for molecular order relative to other silks, allowing the random coil structure to be favored as an adaptation promoting maximal entanglement and adhesion.
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http://dx.doi.org/10.1016/j.ibmb.2013.03.014DOI Listing
July 2013

Natural templates for coiled-coil biomaterials from praying mantis egg cases.

Biomacromolecules 2012 Dec 30;13(12):4264-72. Epub 2012 Nov 30.

Research School of Biology, Australian National University, Canberra, Australia, 0200.

Whereas there is growing interest in producing biomaterials containing coiled-coils, relatively few studies have made use of naturally occurring fibrous proteins. In this study, we have characterized fibrous proteins used by mother praying mantises to produce an extensive covering for their eggs called an ootheca and demonstrate the production of artificial ootheca using recombinantly produced proteins. Examination of natural oothecae by infrared spectroscopy and solid-state nuclear magnetic resonance revealed the material to consist of proteins organized predominately as coiled-coils. Two structural proteins, Mantis Fibroin 1 and Mantis Fibroin 2, were identified in ootheca from each of three species. Between species, the primary sequences of both proteins had diverged considerably, but other features were tightly conserved, including low molecular weight, high abundance of Ala, Glu, Lys, and Ser, and a triblock-like architecture with extensive central coiled-coil domain. Mantis fibroin hydrophobic cores had an unusual composition containing high levels of alanine and aromatic residues. Recombinantly produced mantis fibroins folded into coiled-coils in solution and could be fabricated into solid materials with high coiled-coil content. The structural features of mantis fibroins and their straightforward recombinant production make them promising templates for the production of coiled-coil biomimetics materials.
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http://dx.doi.org/10.1021/bm301570vDOI Listing
December 2012

An evaluation of rheumatology practitioner outreach clinics: a qualitative study.

BMC Health Serv Res 2012 May 20;12:119. Epub 2012 May 20.

Norwich Medical School, University of East Anglia, NR4 7TJ, Norwich, Norfolk, UK.

Background: Services for Rheumatoid Arthritis (RA) have evolved with the development of independently led outreach Rheumatology Practitioner (RP) clinics in Primary Care (PC). Their clinical and cost effectiveness, compared with Secondary Care (SC) services, has not been assessed. The RECIPROCATE study aims to evaluate their clinical and cost effectiveness. This part of the study aimed to explore health professionals' opinions of rheumatology outreach service.

Methods: Using a qualitative design, semi-structured interviews were conducted with GPs, practice nurses, hospital doctors and RPs, from one hospital and seven PC practices in Norfolk, to elicit their opinions of the service. The interviews were analysed using thematic analysis.

Results: All participants agreed the service was supportive and valuable providing high quality personalised care, disease management, social, and educational support. Advantages identified included convenience, continuity of care and proximity of services to home. RPs helped bridge the communication gap between PC and SC. Some participants suggested having a doctor alongside RPs. The service was considered to be cost effective for patients but there was uncertainty about cost effectiveness for service providers. Few disadvantages were identified the most recurring being the lack of other onsite services when needed. It was noted that more services could be provided by RPs such as prescribing and joint injections as well as playing a more active role in knowledge transfer to PC.

Conclusions: Professionals involved in the care of RA patients recognised the valuable role of the RP outreach clinics. This service can be further developed in rheumatology and the example can be replicated for other chronic conditions.
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http://dx.doi.org/10.1186/1472-6963-12-119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457863PMC
May 2012

Silk from crickets: a new twist on spinning.

PLoS One 2012 15;7(2):e30408. Epub 2012 Feb 15.

Evolution, Ecology and Genetics, Research School of Biology, Australian National University, Canberra, Australia.

Raspy crickets (Orthoptera: Gryllacrididae) are unique among the orthopterans in producing silk, which is used to build shelters. This work studied the material composition and the fabrication of cricket silk for the first time. We examined silk-webs produced in captivity, which comprised cylindrical fibers and flat films. Spectra obtained from micro-Raman experiments indicated that the silk is composed of protein, primarily in a beta-sheet conformation, and that fibers and films are almost identical in terms of amino acid composition and secondary structure. The primary sequences of four silk proteins were identified through a mass spectrometry/cDNA library approach. The most abundant silk protein was large in size (300 and 220 kDa variants), rich in alanine, glycine and serine, and contained repetitive sequence motifs; these are features which are shared with several known beta-sheet forming silk proteins. Convergent evolution at the molecular level contrasts with development by crickets of a novel mechanism for silk fabrication. After secretion of cricket silk proteins by the labial glands they are fabricated into mature silk by the labium-hypopharynx, which is modified to allow the controlled formation of either fibers or films. Protein folding into beta-sheet structure during silk fabrication is not driven by shear forces, as is reported for other silks.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0030408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3280245PMC
June 2012

Invited review the coiled coil silk of bees, ants, and hornets.

Biopolymers 2012 Jun 9;97(6):446-54. Epub 2011 Aug 9.

Ecosystem Sciences, Commonwealth Scientific and Research Organisation (CSIRO), Canberra, Australian Capital Territory, Australia.

In this article, we review current knowledge about the silk produced by the larvae of bees, ants, and hornets [Apoidea and Vespoidea: Hymenoptera]. Different species use the silk either alone or in composites for a variety of purposes including mechanical reinforcement, thermal regulation, or humidification. The characteristic molecular structure of this silk is α-helical proteins assembled into tetrameric coiled coils. Gene sequences from seven species are available, and each species possesses a copy of each of four related silk genes that encode proteins predicted to form coiled coils. The proteins are ordered at multiple length scales within the labial gland of the final larval instar before spinning. The insects control the morphology of the silk during spinning to produce either fibers or sheets. The silk proteins are small and non repetitive and have been produced artificially at high levels by fermentation in E. coli. The artificial silk proteins can be fabricated into materials with structural and mechanical properties similar to those of native silks.
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http://dx.doi.org/10.1002/bip.21702DOI Listing
June 2012

Alterations in the Th1/Th2 balance in breast cancer patients using reflexology and scalp massage.

Exp Ther Med 2010 Jan 1;1(1):97-108. Epub 2010 Jan 1.

Postgraduate Medical Institute, University of Hull in association with Hull-York Medical School;

The diagnosis and treatment of breast cancer can adversely affect quality of life. Here the aim was to determine the effects of reflexology on host defences and endocrine function in women with early breast cancer. Six weeks after surgery for early breast cancer, 183 women were randomly assigned to self-initiated support (SIS), SIS plus foot reflexology, or SIS plus scalp massage. Peripheral blood mononuclear cells and serum were isolated at T1 (6 weeks post surgery; baseline), T2 and T3 (4 and 10 weeks post completion of intervention, respectively). Lymphocyte phenotyping found that CD25(+) cells were significantly higher in the massage group compared with the SIS group at T3. The percentage of T cells, and more specifically the T helper subset expressing IL4, decreased significantly in the massage group compared with the SIS group at T3. This change was accompanied by an increase in the percentage of CD8(+) T cytotoxic cells expressing IFNγ in the massage group. Natural killer and lymphokine activated killer cell cytotoxicity measurements, serum levels of cortisol, prolactin and growth hormone, and flow cytometric assessment of their corresponding receptors all revealed no significant differences between the three groups of patients. This study provides evidence that the immunological balance of patients can be altered in a potentially beneficial manner by massage. The original trial was registered with the International Standard Randomised Controlled Trial Registry (ISRCTN87652313).
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http://dx.doi.org/10.3892/etm_00000018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3490382PMC
January 2010

Demographic characteristics of patients using a fully integrated psychosocial support service for cancer patients.

BMC Res Notes 2009 Dec 15;2:253. Epub 2009 Dec 15.

The Institute of Rehabilitation, Hull York Medical School, University of Hull, 215 Anlaby Rd, Kingston upon Hull, East Yorkshire, HU3 2PG, UK.

Background: Psychosocial support services are an important component of modern cancer treatment. A major challenge for all psychosocial services is the achievement of equity of use. Previous studies in the UK have found that women of higher socio-economic status with breast cancer were over-represented amongst those accessing support services. People with other cancer diagnoses, those from socio-economically deprived areas, and men, were under-represented.

Findings: The Oncology Health Service, Kingston Upon Hull, UK, delivers fully integrated psychosocial support and interventions. To assess equity of access in this service, a cross-sectional study of all patients with cancer accessing the service during a 5 day period was carried out. One hundred and forty-five patients attended. Forty four percent were male, and the types of cancer were broadly in the proportions expected on the basis of population prevalence (breast cancer 22%, colorectal cancer 21%, lung cancer 16%). Sixty six percent came from the three most deprived quintiles of the Townsend deprivation Index.

Conclusions: The fully integrated Oncology Health Service in Hull is accessed by a more diverse range of patients than previously reported for other services, and is an example of a model of service by which socially equitable use of psychosocial support in the National Health Service might be achieved.
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http://dx.doi.org/10.1186/1756-0500-2-253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2799437PMC
December 2009

A randomised, controlled trial of the psychological effects of reflexology in early breast cancer.

Eur J Cancer 2010 Jan 10;46(2):312-22. Epub 2009 Nov 10.

The Institute of Rehabilitation, University of Hull, Kingston upon Hull, UK.

Purpose: To conduct a pragmatic randomised controlled trial (RCT) to evaluate the effects of reflexology on quality of life (QofL) in women with early breast cancer.

Patients And Methods: One hundred and eighty-three women were randomised 6 weeks post-breast surgery to self-initiated support (SIS) (comparator intervention), SIS plus reflexology, or SIS plus scalp massage (control for physical and social contact). Reflexology and massage comprised eight sessions at weekly intervals. The primary end-point was 18 weeks post surgery; the primary outcome measure was the Trial Outcome Index (TOI) of the Functional Assessment of Cancer Therapy (FACT-B) - breast cancer version. The secondary end-point was 24 weeks post surgery. Secondary outcome measures were the Hospital Anxiety and Depression Scale (HADS) and the Mood Rating Scale (MRS).

Results: At primary end-point, massage, but not reflexology, was significantly better than SIS on the TOI. Reflexology and massage were both better than SIS for MRS relaxation. Massage was better than reflexology and SIS for MRS easygoingness. At secondary end-point, reflexology, but not massage, was better than SIS on the TOI and MRS relaxation. There were no significant differences between reflexology or massage. There were no significant between group differences in HADS anxiety and depression. Self-reported use of out of study complementary therapies indicated that this was unlikely to have a significant effect on findings.

Conclusions: When compared to SIS, reflexology and massage have statistically significant, and, for reflexology, clinically worthwhile, effects on QofL following surgery for early breast carcinoma.
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http://dx.doi.org/10.1016/j.ejca.2009.10.006DOI Listing
January 2010