Publications by authors named "Jennifer J Smith"

66 Publications

Recent developments in animal venom peptide nanotherapeutics with improved selectivity for cancer cells.

Biotechnol Adv 2021 May 11;50:107769. Epub 2021 May 11.

Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA. Electronic address:

Animal venoms are a rich source of bioactive peptides that efficiently modulate key receptors and ion channels involved in cellular excitability to rapidly neutralize their prey or predators. As such, they have been a wellspring of highly useful pharmacological tools for decades. Besides targeting ion channels, some venom peptides exhibit strong cytotoxic activity and preferentially affect cancer over healthy cells. This is unlikely to be driven by an evolutionary impetus, and differences in tumor cells and the tumor microenvironment are probably behind the serendipitous selectivity shown by some venom peptides. However, strategies such as bioconjugation and nanotechnologies are showing potential to improve their selectivity and potency, thereby paving the way to efficiently harness new anticancer mechanisms offered by venom peptides. This review aims to highlight advances in nano- and chemotherapeutic tools and prospective anti-cancer drug leads derived from animal venom peptides.
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http://dx.doi.org/10.1016/j.biotechadv.2021.107769DOI Listing
May 2021

A Peer-Based Strategy to Overcome HPV Vaccination Inequities in Rural Communities: A Physical Distancing-Compliant Approach.

Crit Rev Eukaryot Gene Expr 2021 ;31(1):61-69

KM Consulting, New Jersey, USA.

The human papilloma virus (HPV) vaccine is the world's first proven and effective vaccine to prevent cancers in males and females when administered pre-exposure. Like most of the US, barely half of Vermont teens are up-to-date with the vaccination, with comparable deficits in New Hampshire and Maine. The rates for HPV vaccine initiation and completion are as low as 33% in rural New England. Consequently, there is a compelling responsibility to communicate its importance to unvaccinated teenagers before their risk for infection increases. Messaging in rural areas promoting HPV vaccination is compromised by community-based characteristics that include access to appropriate medical care, poor media coverage, parental and peer influence, and skepticism of science and medicine. Current strategies are predominantly passive access to literature and Internet-based information. Evidence indicates that performance-based messaging can clarify the importance of HPV vaccination to teenagers and their parents in rural areas. Increased HPV vaccination will significantly contribute to the prevention of a broadening spectrum of cancers. Reducing rurality-based inequities is a public health priority. Development of a performance-based peer-communication intervention can capture a window of opportunity to provide increasingly effective and sustained HPV protection. An effective approach can be partnering rural schools and regional health teams with a program that is nimble and scalable to respond to public health policies and practices compliant with COVID-19 pandemic-related modifications on physical distancing and interacting in the foreseeable future.
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http://dx.doi.org/10.1615/CritRevEukaryotGeneExpr.2021036945DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128079PMC
March 2021

Innovative Approaches to Emergency Medical Services Fellowship Challenges.

West J Emerg Med 2020 Feb 21;21(2):429-433. Epub 2020 Feb 21.

Medical College of Wisconsin, Department of Emergency Medicine, Milwaukee, Wisconsin.

Introduction: Since the development of an Accreditation Council of Graduate Medical Education (ACGME)-accredited emergency medical services (EMS) fellowship, there has been little published literature on effective methods of content delivery or training modalities. Here we explore a variety of innovative approaches to the development and revision of the EMS fellowship curriculum.

Methods: Three academic, university-based ACGME-accredited EMS fellowship programs each implemented an innovative change to their existing training curricula. These changes included the following: a novel didactic curriculum delivery modality and evaluation; implementation of a distance education program to improve EMS fellows' rural EMS experiences; and modification of an existing EMS fellowship curriculum to train a non-emergency medicine physician.

Results: Changes made to each of the above EMS fellowship programs addressed unique challenges, demonstrating areas of success and promise for more generalized implementation of these curricula. Obstacles remain in tailoring the described curricula to the needs of each unique institution and system.

Conclusion: Three separate curricula and program changes were implemented to overcome specific challenges and achieve educational goals. It is our hope that our shared experiences will enable others in addressing common barriers to teaching the EMS fellowship core content and share similar innovative approaches to educational challenges.
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http://dx.doi.org/10.5811/westjem.2019.10.43830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081868PMC
February 2020

GluClR-mediated inhibitory postsynaptic currents reveal targets for ivermectin and potential mechanisms of ivermectin resistance.

PLoS Pathog 2019 01 29;15(1):e1007570. Epub 2019 Jan 29.

Queensland Brain Institute, The University of Queensland, Brisbane, Australia.

Glutamate-gated chloride channel receptors (GluClRs) mediate inhibitory neurotransmission at invertebrate synapses and are primary targets of parasites that impact drastically on agriculture and human health. Ivermectin (IVM) is a broad-spectrum pesticide that binds and potentiates GluClR activity. Resistance to IVM is a major economic and health concern, but the molecular and synaptic mechanisms of resistance are ill-defined. Here we focus on GluClRs of the agricultural endoparasite, Haemonchus contortus. We demonstrate that IVM potentiates inhibitory input by inducing a tonic current that plateaus over 15 minutes and by enhancing post-synaptic current peak amplitude and decay times. We further demonstrate that IVM greatly enhances the active durations of single receptors. These effects are greatly attenuated when endogenous IVM-insensitive subunits are incorporated into GluClRs, suggesting a mechanism of IVM resistance that does not affect glutamate sensitivity. We discovered functional groups of IVM that contribute to tuning its potency at different isoforms and show that the dominant mode of access of IVM is via the cell membrane to the receptor.
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http://dx.doi.org/10.1371/journal.ppat.1007570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368337PMC
January 2019

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold.

Cell Mol Life Sci 2018 Dec 14;75(24):4511-4524. Epub 2018 Aug 14.

Centre for Advanced Imaging, The University of Queensland, St Lucia, QLD, 4072, Australia.

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type K channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.
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http://dx.doi.org/10.1007/s00018-018-2897-6DOI Listing
December 2018

Structural basis for the modulation of voltage-gated sodium channels by animal toxins.

Science 2018 10 26;362(6412). Epub 2018 Jul 26.

State Key Laboratory of Membrane Biology, School of Life Sciences and School of Medicine, Tsinghua University, Beijing 100084, China.

Animal toxins that modulate the activity of voltage-gated sodium (Na) channels are broadly divided into two categories-pore blockers and gating modifiers. The pore blockers tetrodotoxin (TTX) and saxitoxin (STX) are responsible for puffer fish and shellfish poisoning in humans, respectively. Here, we present structures of the insect Na channel NaPaS bound to a gating modifier toxin Dc1a at 2.8 angstrom-resolution and in the presence of TTX or STX at 2.6-Å and 3.2-Å resolution, respectively. Dc1a inserts into the cleft between VSD and the pore of NaPaS, making key contacts with both domains. The structures with bound TTX or STX reveal the molecular details for the specific blockade of Na access to the selectivity filter from the extracellular side by these guanidinium toxins. The structures shed light on structure-based development of Na channel drugs.
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http://dx.doi.org/10.1126/science.aau2596DOI Listing
October 2018

True Lies: Using Proteomics to Assess the Accuracy of Transcriptome-Based Venomics in Centipedes Uncovers False Positives and Reveals Startling Intraspecific Variation in Scolopendra Subspinipes.

Toxins (Basel) 2018 02 28;10(3). Epub 2018 Feb 28.

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

Centipede venoms have emerged as a rich source of novel bioactive compounds. However, most centipede species are commonly considered too small for venom extraction and transcriptomics is likely to be an attractive way of probing the molecular diversity of these venoms. Examining the venom composition of , we test the accuracy of this approach. We compared the proteomically determined venom profile with four common toxin transcriptomic toxin annotation approaches: BLAST search against toxins in UniProt, lineage-specific toxins, or species-specific toxins and comparative expression analyses of venom and non-venom producing tissues. This demonstrated that even toxin annotation based on lineage-specific homology searches is prone to substantial errors compared to a proteomic approach. However, combined comparative transcriptomics and phylogenetic analysis of putative toxin families substantially improves annotation accuracy. Furthermore, comparison of the venom composition of with the closely related revealed a surprising lack of overlap. This first insight into the intraspecific venom variability of centipedes contrasts the sequence conservation expected from previous findings that centipede toxins evolve under strong negative selection. Our results highlight the importance of proteomic data in studies of even comparably well-characterized venoms and warrants caution when sourcing venom from centipedes of unknown origin.
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http://dx.doi.org/10.3390/toxins10030096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5869384PMC
February 2018

The Aromatic Head Group of Spider Toxin Polyamines Influences Toxicity to Cancer Cells.

Toxins (Basel) 2017 10 27;9(11). Epub 2017 Oct 27.

Centre for Biodiscovery and Molecular Development of Therapeutics, AITHM, James Cook University, Cairns, QLD 4878, Australia.

Spider venoms constitute incredibly diverse libraries of compounds, many of which are involved in prey capture and defence. Polyamines are often prevalent in the venom and target ionotropic glutamate receptors. Here we show that a novel spider polyamine, PA, containing a hydroxyphenyl-based structure is present in the venom of several species of tarantula, and has selective toxicity against MCF-7 breast cancer cells. By contrast, a polyamine from an Australian funnel-web spider venom, which contains an identical polyamine tail to PA but an indole-based head-group, is only cytotoxic at high concentrations. Our results suggest that the ring structure plays a role in the cytotoxicity and that modification to the polyamine head group might lead to more potent and selective compounds with potential as novel cancer treatments.
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http://dx.doi.org/10.3390/toxins9110346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705961PMC
October 2017

Adaptive Prediction Emerges Over Short Evolutionary Time Scales.

Genome Biol Evol 2017 06;9(6):1616-1623

Institute for Systems Biology, Seattle, Washington.

Adaptive prediction is a capability of diverse organisms, including microbes, to sense a cue and prepare in advance to deal with a future environmental challenge. Here, we investigated the timeframe over which adaptive prediction emerges when an organism encounters an environment with novel structure. We subjected yeast to laboratory evolution in a novel environment with repetitive, coupled exposures to a neutral chemical cue (caffeine), followed by a sublethal dose of a toxin (5-FOA), with an interspersed requirement for uracil prototrophy to counter-select mutants that gained constitutive 5-FOA resistance. We demonstrate the remarkable ability of yeast to internalize a novel environmental pattern within 50-150 generations by adaptively predicting 5-FOA stress upon sensing caffeine. We also demonstrate how novel environmental structure can be internalized by coupling two unrelated response networks, such as the response to caffeine and signaling-mediated conditional peroxisomal localization of proteins.
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http://dx.doi.org/10.1093/gbe/evx116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570091PMC
June 2017

Evidence that auxin is required for normal seed size and starch synthesis in pea.

New Phytol 2017 Oct 27;216(1):193-204. Epub 2017 Jul 27.

School of Biological Sciences, University of Tasmania, Sandy Bay, 7001, Australia.

In recent years the biosynthesis of auxin has been clarified with the aid of mutations in auxin biosynthesis genes. However, we know little about the effects of these mutations on the seed-filling stage of seed development. Here we investigate a key auxin biosynthesis mutation of the garden pea, which results in auxin deficiency in developing seeds. We exploit the large seed size of this model species, which facilitates the measurement of compounds in individual seeds. The mutation results in small seeds with reduced starch content and a wrinkled phenotype at the dry stage. The phenotypic effects of the mutation were fully reversed by introduction of the wild-type gene as a transgene, and partially reversed by auxin application. The results indicate that auxin is required for normal seed size and starch accumulation in pea, an important grain legume crop.
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http://dx.doi.org/10.1111/nph.14690DOI Listing
October 2017

An indicator cell assay for blood-based diagnostics.

PLoS One 2017 8;12(6):e0178608. Epub 2017 Jun 8.

Institute for Systems Biology, Seattle, WA, United States of America.

We have established proof of principle for the Indicator Cell Assay Platform™ (iCAP™), a broadly applicable tool for blood-based diagnostics that uses specifically-selected, standardized cells as biosensors, relying on their innate ability to integrate and respond to diverse signals present in patients' blood. To develop an assay, indicator cells are exposed in vitro to serum from case or control subjects and their global differential response patterns are used to train reliable, disease classifiers based on a small number of features. In a feasibility study, the iCAP detected pre-symptomatic disease in a murine model of amyotrophic lateral sclerosis (ALS) with 94% accuracy (p-Value = 3.81E-6) and correctly identified samples from a murine Huntington's disease model as non-carriers of ALS. Beyond the mouse model, in a preliminary human disease study, the iCAP detected early stage Alzheimer's disease with 72% cross-validated accuracy (p-Value = 3.10E-3). For both assays, iCAP features were enriched for disease-related genes, supporting the assay's relevance for disease research.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178608PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464608PMC
September 2017

Modulatory features of the novel spider toxin μ-TRTX-Df1a isolated from the venom of the spider Davus fasciatus.

Br J Pharmacol 2017 08 27;174(15):2528-2544. Epub 2017 Jun 27.

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

Background And Purpose: Naturally occurring dysfunction of voltage-gated sodium (Na ) channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel Na modulators, we investigated spider venoms for new inhibitors of Na channels.

Experimental Approach: We used high-throughput screens to identify a Na modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modelling and a rodent pain model.

Key Results: We identified a potent Na inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by Na 1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited voltage-gated calcium (Ca 3) currents but had no activity against the voltage-gated potassium (K 2) channel. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the Na channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all Na subtypes tested (hNa 1.1-1.7). Df1a also slowed fast inactivation of Na 1.1, Na 1.3 and Na 1.5 and modified the voltage-dependence of activation and inactivation of most of the Na subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which probably explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviours induced by the Na activator OD1.

Conclusion And Implications: μ-TRTX-Df1a shows potential as a new molecule for the development of drugs to treat pain disorders mediated by voltage-gated ion channels.
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http://dx.doi.org/10.1111/bph.13865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5513869PMC
August 2017

Combining inferred regulatory and reconstructed metabolic networks enhances phenotype prediction in yeast.

PLoS Comput Biol 2017 05 17;13(5):e1005489. Epub 2017 May 17.

Institute for Systems Biology, Seattle, Washington, United States of America.

Gene regulatory and metabolic network models have been used successfully in many organisms, but inherent differences between them make networks difficult to integrate. Probabilistic Regulation Of Metabolism (PROM) provides a partial solution, but it does not incorporate network inference and underperforms in eukaryotes. We present an Integrated Deduced And Metabolism (IDREAM) method that combines statistically inferred Environment and Gene Regulatory Influence Network (EGRIN) models with the PROM framework to create enhanced metabolic-regulatory network models. We used IDREAM to predict phenotypes and genetic interactions between transcription factors and genes encoding metabolic activities in the eukaryote, Saccharomyces cerevisiae. IDREAM models contain many fewer interactions than PROM and yet produce significantly more accurate growth predictions. IDREAM consistently outperformed PROM using any of three popular yeast metabolic models and across three experimental growth conditions. Importantly, IDREAM's enhanced accuracy makes it possible to identify subtle synthetic growth defects. With experimental validation, these novel genetic interactions involving the pyruvate dehydrogenase complex suggested a new role for fatty acid-responsive factor Oaf1 in regulating acetyl-CoA production in glucose grown cells.
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http://dx.doi.org/10.1371/journal.pcbi.1005489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453602PMC
May 2017

Body Temperature after EMS Transport: Association with Traumatic Brain Injury Outcomes.

Prehosp Emerg Care 2017 Sep-Oct;21(5):575-582. Epub 2017 May 8.

Introduction: Low body temperatures following prehospital transport are associated with poor outcomes in patients with traumatic brain injury (TBI). However, a minimal amount is known about potential associations across a range of temperatures obtained immediately after prehospital transport. Furthermore, a minimal amount is known about the influence of body temperature on non-mortality outcomes. The purpose of this study was to assess the correlation between temperatures obtained immediately following prehospital transport and TBI outcomes across the entire range of temperatures.

Methods: This retrospective observational study included all moderate/severe TBI cases (CDC Barell Matrix Type 1) in the pre-implementation cohort of the Excellence in Prehospital Injury Care (EPIC) TBI Study (NIH/NINDS: 1R01NS071049). Cases were compared across four cohorts of initial trauma center temperature (ITCT): <35.0°C [Very Low Temperature (VLT)]; 35.0-35.9°C [Low Temperature (LT)]; 36.0-37.9°C [Normal Temperature (NT)]; and ≥38.0°C [Elevated Temperature (ET)]. Multivariable analysis was performed adjusting for injury severity score, age, sex, race, ethnicity, blunt/penetrating trauma, and payment source. Adjusted odds ratios (aORs) with 95% confidence intervals (CI) for mortality were calculated. To evaluate non-mortality outcomes, deaths were excluded and the adjusted median increase in hospital length of stay (LOS), ICU LOS and total hospital charges were calculated for each ITCT group and compared to the NT group.

Results: 22,925 cases were identified and cases with interfacility transfer (7361, 32%), no EMS transport (1213, 5%), missing ITCT (2083, 9%), or missing demographic data (391, 2%) were excluded. Within this study cohort the aORs for death (compared to the NT group) were 2.41 (CI: 1.83-3.17) for VLT, 1.62 (CI: 1.37-1.93) for LT, and 1.86 (CI: 1.52-3.00) for ET. Similarly, trauma center (TC) LOS, ICU LOS, and total TC charges increased in all temperature groups when compared to NT.

Conclusion: In this large, statewide study of major TBI, both ETs and LTs immediately following prehospital transport were independently associated with higher mortality and with increased TC LOS, ICU LOS, and total TC charges. Further study is needed to identify the causes of abnormal body temperature during the prehospital interval and if in-field measures to prevent temperature variations might improve outcomes.
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http://dx.doi.org/10.1080/10903127.2017.1308609DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638643PMC
July 2018

Insect-Active Toxins with Promiscuous Pharmacology from the African Theraphosid Spider Monocentropus balfouri.

Toxins (Basel) 2017 05 5;9(5). Epub 2017 May 5.

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

Many chemical insecticides are becoming less efficacious due to rising resistance in pest species, which has created much interest in the development of new, eco-friendly bioinsecticides. Since insects are the primary prey of most spiders, their venoms are a rich source of insect-active peptides that can be used as leads for new bioinsecticides or as tools to study molecular receptors that are insecticidal targets. In the present study, we isolated two insecticidal peptides, µ/ω-TRTX-Mb1a and -Mb1b, from venom of the African tarantula Recombinant µ/ω-TRTX-Mb1a and -Mb1b paralyzed both (Australian sheep blowfly) and (housefly), but neither peptide affected larvae of (cotton bollworms). Both peptides inhibited currents mediated by voltage-gated sodium (Na) and calcium channels in (American cockroach) dorsal unpaired median neurons, and they also inhibited the cloned (German cockroach) Na channel (BgNa1). An additional effect seen only with Mb1a on BgNa1 was a delay in fast inactivation. Comparison of the Na channel sequences of the tested insect species revealed that variations in the S1-S2 loops in the voltage sensor domains might underlie the differences in activity between different phyla.
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http://dx.doi.org/10.3390/toxins9050155DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450703PMC
May 2017

Pharmacological characterisation of the highly Na1.7 selective spider venom peptide Pn3a.

Sci Rep 2017 01 20;7:40883. Epub 2017 Jan 20.

IMB Centre for Pain Research, Institute for Molecular Bioscience, 306 Carmody Rd (Building 80), The University of Queensland, St Lucia, Queensland, 4072, Australia.

Human genetic studies have implicated the voltage-gated sodium channel Na1.7 as a therapeutic target for the treatment of pain. A novel peptide, μ-theraphotoxin-Pn3a, isolated from venom of the tarantula Pamphobeteus nigricolor, potently inhibits Na1.7 (IC 0.9 nM) with at least 40-1000-fold selectivity over all other Na subtypes. Despite on-target activity in small-diameter dorsal root ganglia, spinal slices, and in a mouse model of pain induced by Na1.7 activation, Pn3a alone displayed no analgesic activity in formalin-, carrageenan- or FCA-induced pain in rodents when administered systemically. A broad lack of analgesic activity was also found for the selective Na1.7 inhibitors PF-04856264 and phlotoxin 1. However, when administered with subtherapeutic doses of opioids or the enkephalinase inhibitor thiorphan, these subtype-selective Na1.7 inhibitors produced profound analgesia. Our results suggest that in these inflammatory models, acute administration of peripherally restricted Na1.7 inhibitors can only produce analgesia when administered in combination with an opioid.
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http://dx.doi.org/10.1038/srep40883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5247677PMC
January 2017

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

G3 (Bethesda) 2017 01 5;7(1):279-288. Epub 2017 Jan 5.

Institute for Systems Biology, Seattle, Washington 98109

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAY extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.
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http://dx.doi.org/10.1534/g3.116.037044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217116PMC
January 2017

Isolation of two insecticidal toxins from venom of the Australian theraphosid spider Coremiocnemis tropix.

Toxicon 2016 Dec 26;123:62-70. Epub 2016 Oct 26.

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

Sheep flystrike is caused by parasitic flies laying eggs on soiled wool or open wounds, after which the hatched maggots feed on the sheep flesh and often cause large lesions. It is a significant economic problem for the livestock industry as infestations are difficult to control due to ongoing cycles of larval development into flies followed by further egg laying. We therefore screened venom fractions from the Australian theraphosid spider Coremiocnemis tropix to identify toxins active against the sheep blowfly Lucilia cuprina, which is the primary cause of flystrike in Australia. This screen led to isolation of two insecticidal peptides, Ct1a and Ct1b, that are lethal to blowflies within 24 h of injection. The primary structure of these peptides was determined using a combination of Edman degradation and sequencing of a C. tropix venom-gland transcriptome. Ct1a and Ct1b contain 39 and 38 amino acid residues, respectively, including six cysteine residues that form three disulfide bonds. Recombinant production in bacteria (Escherichia coli) resulted in low yields of Ct1a whereas solid-phase peptide synthesis using native chemical ligation produced sufficient quantities of Ct1a for functional analyses. Synthetic Ct1a had no effect on voltage-gated sodium channels from the American cockroach Periplanata americana or the German cockroach Blattella germanica, but it was lethal to sheep blowflies with an LD of 1687 pmol/g.
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http://dx.doi.org/10.1016/j.toxicon.2016.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5881108PMC
December 2016

Molecular basis of the remarkable species selectivity of an insecticidal sodium channel toxin from the African spider Augacephalus ezendami.

Sci Rep 2016 07 7;6:29538. Epub 2016 Jul 7.

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

The inexorable decline in the armament of registered chemical insecticides has stimulated research into environmentally-friendly alternatives. Insecticidal spider-venom peptides are promising candidates for bioinsecticide development but it is challenging to find peptides that are specific for targeted pests. In the present study, we isolated an insecticidal peptide (Ae1a) from venom of the African spider Augacephalus ezendami (family Theraphosidae). Injection of Ae1a into sheep blowflies (Lucilia cuprina) induced rapid but reversible paralysis. In striking contrast, Ae1a was lethal to closely related fruit flies (Drosophila melanogaster) but induced no adverse effects in the recalcitrant lepidopteran pest Helicoverpa armigera. Electrophysiological experiments revealed that Ae1a potently inhibits the voltage-gated sodium channel BgNaV1 from the German cockroach Blattella germanica by shifting the threshold for channel activation to more depolarized potentials. In contrast, Ae1a failed to significantly affect sodium currents in dorsal unpaired median neurons from the American cockroach Periplaneta americana. We show that Ae1a interacts with the domain II voltage sensor and that sensitivity to the toxin is conferred by natural sequence variations in the S1-S2 loop of domain II. The phyletic specificity of Ae1a provides crucial information for development of sodium channel insecticides that target key insect pests without harming beneficial species.
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http://dx.doi.org/10.1038/srep29538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935840PMC
July 2016

Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating.

Biochim Biophys Acta 2016 Apr 2;1858(4):872-82. Epub 2016 Feb 2.

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

Many venom peptides are potent and selective inhibitors of voltage-gated ion channels, including channels that are validated therapeutic targets for treatment of a wide range of human diseases. However, the development of novel venom-peptide-based therapeutics requires an understanding of their mechanism of action. In the case of voltage-gated ion channels, venom peptides act either as pore blockers that bind to the extracellular side of the channel pore or gating modifiers that bind to one or more of the membrane-embedded voltage sensor domains. In the case of gating modifiers, it has been debated whether the peptide must partition into the membrane to reach its binding site. In this study, we used surface plasmon resonance, fluorescence spectroscopy and molecular dynamics to directly compare the lipid-binding properties of two gating modifiers (μ-TRTX-Hd1a and ProTx-I) and two pore blockers (ShK and KIIIA). Only ProTx-I was found to bind to model membranes. Our results provide further evidence that the ability to insert into the lipid bilayer is not a requirement to be a gating modifier. In addition, we characterised the surface of ProTx-I that mediates its interaction with neutral and anionic phospholipid membranes and show that it preferentially interacts with anionic lipids.
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http://dx.doi.org/10.1016/j.bbamem.2016.02.002DOI Listing
April 2016

δ-Conotoxin SuVIA suggests an evolutionary link between ancestral predator defence and the origin of fish-hunting behaviour in carnivorous cone snails.

Proc Biol Sci 2015 Jul;282(1811)

The Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia Institut des Biomolécules Max Mousseron, UMR 5247, Université Montpellier - CNRS, Place Eugène Bataillon, Montpellier Cedex 5 34095, France

Some venomous cone snails feed on small fishes using an immobilizing combination of synergistic venom peptides that target Kv and Nav channels. As part of this envenomation strategy, δ-conotoxins are potent ichtyotoxins that enhance Nav channel function. δ-Conotoxins belong to an ancient and widely distributed gene superfamily, but any evolutionary link from ancestral worm-eating cone snails to modern piscivorous species has not been elucidated. Here, we report the discovery of SuVIA, a potent vertebrate-active δ-conotoxin characterized from a vermivorous cone snail (Conus suturatus). SuVIA is equipotent at hNaV1.3, hNaV1.4 and hNaV1.6 with EC50s in the low nanomolar range. SuVIA also increased peak hNaV1.7 current by approximately 75% and shifted the voltage-dependence of activation to more hyperpolarized potentials from -15 mV to -25 mV, with little effect on the voltage-dependence of inactivation. Interestingly, the proximal venom gland expression and pain-inducing effect of SuVIA in mammals suggest that δ-conotoxins in vermivorous cone snails play a defensive role against higher order vertebrates. We propose that δ-conotoxins originally evolved in ancestral vermivorous cones to defend against larger predators including fishes have been repurposed to facilitate a shift to piscivorous behaviour, suggesting an unexpected underlying mechanism for this remarkable evolutionary transition.
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http://dx.doi.org/10.1098/rspb.2015.0817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4528551PMC
July 2015

Bicluster Sampled Coherence Metric (BSCM) provides an accurate environmental context for phenotype predictions.

BMC Syst Biol 2015 15;9 Suppl 2:S1. Epub 2015 Apr 15.

Background: Biclustering is a popular method for identifying under which experimental conditions biological signatures are co-expressed. However, the general biclustering problem is NP-hard, offering room to focus algorithms on specific biological tasks. We hypothesize that conditional co-regulation of genes is a key factor in determining cell phenotype and that accurately segregating conditions in biclusters will improve such predictions. Thus, we developed a bicluster sampled coherence metric (BSCM) for determining which conditions and signals should be included in a bicluster.

Results: Our BSCM calculates condition and cluster size specific p-values, and we incorporated these into the popular integrated biclustering algorithm cMonkey. We demonstrate that incorporation of our new algorithm significantly improves bicluster co-regulation scores (p-value = 0.009) and GO annotation scores (p-value = 0.004). Additionally, we used a bicluster based signal to predict whether a given experimental condition will result in yeast peroxisome induction. Using the new algorithm, the classifier accuracy improves from 41.9% to 76.1% correct.

Conclusions: We demonstrate that the proposed BSCM helps determine which signals ought to be co-clustered, resulting in more accurately assigned bicluster membership. Furthermore, we show that BSCM can be extended to more accurately detect under which experimental conditions the genes are co-clustered. Features derived from this more accurate analysis of conditional regulation results in a dramatic improvement in the ability to predict a cellular phenotype in yeast. The latest cMonkey is available for download at https://github.com/baliga-lab/cmonkey2. The experimental data and source code featured in this paper is available http://AitchisonLab.com/BSCM. BSCM has been incorporated in the official cMonkey release.
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http://dx.doi.org/10.1186/1752-0509-9-S2-S1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4407105PMC
February 2016

Seven novel modulators of the analgesic target NaV 1.7 uncovered using a high-throughput venom-based discovery approach.

Br J Pharmacol 2015 May 4;172(10):2445-58. Epub 2015 Mar 4.

Centre for Pain Research, Institute for Molecular Bioscience, St. Lucia, Qld, Australia.

Background And Purpose: Chronic pain is a serious worldwide health issue, with current analgesics having limited efficacy and dose-limiting side effects. Humans with loss-of-function mutations in the voltage-gated sodium channel NaV 1.7 (hNaV 1.7) are indifferent to pain, making hNaV 1.7 a promising target for analgesic development. Since spider venoms are replete with NaV channel modulators, we examined their potential as a source of hNaV 1.7 inhibitors.

Experimental Approach: We developed a high-throughput fluorescent-based assay to screen spider venoms against hNaV 1.7 and isolate 'hit' peptides. To examine the binding site of these peptides, we constructed a panel of chimeric channels in which the S3b-S4 paddle motif from each voltage sensor domain of hNaV 1.7 was transplanted into the homotetrameric KV 2.1 channel.

Key Results: We screened 205 spider venoms and found that 40% contain at least one inhibitor of hNaV 1.7. By deconvoluting 'hit' venoms, we discovered seven novel members of the NaSpTx family 1. One of these peptides, Hd1a (peptide μ-TRTX-Hd1a from venom of the spider Haplopelma doriae), inhibited hNaV 1.7 with a high level of selectivity over all other subtypes, except hNaV 1.1. We showed that Hd1a is a gating modifier that inhibits hNaV 1.7 by interacting with the S3b-S4 paddle motif in channel domain II. The structure of Hd1a, determined using heteronuclear NMR, contains an inhibitor cystine knot motif that is likely to confer high levels of chemical, thermal and biological stability.

Conclusion And Implications: Our data indicate that spider venoms are a rich natural source of hNaV 1.7 inhibitors that might be useful leads for the development of novel analgesics.
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http://dx.doi.org/10.1111/bph.13081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409898PMC
May 2015

Peroxisomes take shape.

Nat Rev Mol Cell Biol 2013 Dec;14(12):803-17

1] Seattle Biomedical Research Institute, 307 Westlake Avenue North, 98109-5240, USA. [2] Institute for Systems Biology, 401 Terry Avenue North, Seattle, Washington 98109-5219, USA.

Peroxisomes carry out various oxidative reactions that are tightly regulated to adapt to the changing needs of the cell and varying external environments. Accordingly, they are remarkably fluid and can change dramatically in abundance, size, shape and content in response to numerous cues. These dynamics are controlled by multiple aspects of peroxisome biogenesis that are coordinately regulated with each other and with other cellular processes. Ongoing studies are deciphering the diverse molecular mechanisms that underlie biogenesis and how they cooperate to dynamically control peroxisome utility. These important challenges should lead to an understanding of peroxisome dynamics that can be capitalized upon for bioengineering and the development of therapies to improve human health.
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http://dx.doi.org/10.1038/nrm3700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4060825PMC
December 2013

Molecular mechanisms of system responses to novel stimuli are predictable from public data.

Nucleic Acids Res 2014 Feb 31;42(3):1442-60. Epub 2013 Oct 31.

Seattle Biomedical Research Institute, Seattle, WA 98109-5219 USA, Institute for Systems Biology, Seattle, WA 98109-5240 USA, The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Science, Southeast University, Nanjing 210096, China and Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan 704, Taiwan.

Systems scale models provide the foundation for an effective iterative cycle between hypothesis generation, experiment and model refinement. Such models also enable predictions facilitating the understanding of biological complexity and the control of biological systems. Here, we demonstrate the reconstruction of a globally predictive gene regulatory model from public data: a model that can drive rational experiment design and reveal new regulatory mechanisms underlying responses to novel environments. Specifically, using ∼ 1500 publically available genome-wide transcriptome data sets from Saccharomyces cerevisiae, we have reconstructed an environment and gene regulatory influence network that accurately predicts regulatory mechanisms and gene expression changes on exposure of cells to completely novel environments. Focusing on transcriptional networks that induce peroxisomes biogenesis, the model-guided experiments allow us to expand a core regulatory network to include novel transcriptional influences and linkage across signaling and transcription. Thus, the approach and model provides a multi-scalar picture of gene dynamics and are powerful resources for exploiting extant data to rationally guide experimentation. The techniques outlined here are generally applicable to any biological system, which is especially important when experimental systems are challenging and samples are difficult and expensive to obtain-a common problem in laboratory animal and human studies.
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http://dx.doi.org/10.1093/nar/gkt938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919619PMC
February 2014

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

Proc Natl Acad Sci U S A 2013 May 13;110(22):8906-11. Epub 2013 May 13.

Chemical and Structural Biology, Institute for Molecular Biosciences, University of Queensland, St. Lucia, QLD 4072, Australia.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.
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http://dx.doi.org/10.1073/pnas.1214062110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670328PMC
May 2013

The insecticidal potential of venom peptides.

Cell Mol Life Sci 2013 Oct 23;70(19):3665-93. Epub 2013 Mar 23.

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

Pest insect species are a burden to humans as they destroy crops and serve as vectors for a wide range of diseases including malaria and dengue. Chemical insecticides are currently the dominant approach for combating these pests. However, the de-registration of key classes of chemical insecticides due to their perceived ecological and human health risks in combination with the development of insecticide resistance in many pest insect populations has created an urgent need for improved methods of insect pest control. The venoms of arthropod predators such as spiders and scorpions are a promising source of novel insecticidal peptides that often have different modes of action to extant chemical insecticides. These peptides have been optimized via a prey-predator arms race spanning hundreds of millions of years to target specific types of insect ion channels and receptors. Here we review the current literature on insecticidal venom peptides, with a particular focus on their structural and pharmacological diversity, and discuss their potential for deployment as insecticides.
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http://dx.doi.org/10.1007/s00018-013-1315-3DOI Listing
October 2013

Global analysis of condition-specific subcellular protein distribution and abundance.

Mol Cell Proteomics 2013 May 24;12(5):1421-35. Epub 2013 Jan 24.

Molecular and Cellular Biology Program, University of Washington, Seattle, Washington 98195, USA.

Cellular control of protein activities by modulation of their abundance or compartmentalization is not easily measured on a large scale. We developed and applied a method to globally interrogate these processes that is widely useful for systems-level analyses of dynamic cellular responses in many cell types. The approach involves subcellular fractionation followed by comprehensive proteomic analysis of the fractions, which is enabled by a data-independent acquisition mass spectrometry approach that samples every available mass to charge channel systematically to maximize sensitivity. Next, various fraction-enrichment ratios are measured for all detected proteins across different environmental conditions and used to group proteins into clusters reflecting changes in compartmentalization and relative conditional abundance. Application of the approach to characterize the response of yeast proteins to fatty acid exposure revealed dynamics of peroxisomes and novel dynamics of MCC/eisosomes, specialized plasma membrane domains comprised of membrane compartment occupied by Can1 (MCC) and eisosome subdomains. It also led to the identification of Fat3, a fatty acid transport protein of the plasma membrane, previously annotated as Ykl187.
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http://dx.doi.org/10.1074/mcp.O112.019166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650349PMC
May 2013

Mass landscapes of seven scorpion species: The first analyses of Australian species with 1,5-DAN matrix.

J Venom Res 2012 23;3:7-14. Epub 2012 Oct 23.

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

Scorpion venoms have been studied for over fifty years; however, the majority of research has focussed primarily on medically important Buthidae species. Additionally, venoms of the estimated 200 species of scorpion native to Australia have received very little attention. The first venom mass profiles of six non-buthid and one buthid scorpion species are presented herein, four of which are endemic to Australia. While masses under 5 kDa dominated the venoms of all species, the buthid venom contained considerably more masses between 7 and 8 kDa than those of the non-buthids, corroborating the emergent trend that buthids are richer in long-chain neurotoxins than non-buthids. The Australian scorpion venom fractions were also analysed with the relatively new MALDI-ToF matrix 1,5-DAN. Over forty partial sequences were obtained, the majority of which are homologous to scorpion antimicrobials such as opistoporin and IsCT2. Overall, this study is the single most comprehensive mass spectrometric analysis of scorpion venom landscapes to date and provides an insight into untapped Australian species.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518322PMC
December 2012