Crown-of-thorns starfish spines secrete defence proteins.

Background: The crown-of-thorns starfish (COTS; Acanthaster species) is a slow-moving corallivore protected by an extensive array of long, sharp toxic spines. Envenomation can result in nausea, numbness, vomiting, joint aches and sometimes paralysis. Small molecule saponins and the plancitoxin proteins have been implicated in COTS toxicity. Methods: Brine shrimp lethality assays were used to confirm the secretion of spine toxin biomolecules. Histological analysis, followed by spine-derived proteomics helped to explain the source and identity of proteins, while quantitative RNA-sequencing and phylogeny confirmed target gene expression and relative conservation, respectively. Results: We demonstrate the lethality of COTS spine secreted biomolecules on brine shrimp, including significant toxicity using aboral spine semi-purifications of >10 kDa (p > 0.05, 9.82 µg/ml), supporting the presence of secreted proteins as toxins. Ultrastructure observations of the COTS aboral spine showed the presence of pores that could facilitate the distribution of secreted proteins. Subsequent purification and mass spectrometry analysis of spine-derived proteins identified numerous secretory proteins, including plancitoxins, as well as those with relatively high gene expression in spines, including phospholipase A2, protease inhibitor 16-like protein, ependymin-related proteins and those uncharacterized. Some secretory proteins (e.g., vitellogenin and deleted in malignant brain tumor protein 1) were not highly expressed in spine tissue, yet the spine may serve as a storage or release site. This study contributes to our understanding of the COTS through functional, ultrastructural and proteomic analysis of aboral spines.

Quantitative proteomic landscape of unstable atherosclerosis identifies molecular signatures and therapeutic targets for plaque stabilization.

Atherosclerotic plaque rupture leading to myocardial infarction is a major global health burden. Applying the tandem stenosis (TS) mouse model, which distinctively exhibits the characteristics of human plaque instability/rupture, we use quantitative proteomics to understand and directly compare unstable and stable atherosclerosis. Our data highlight the disparate natures and define unique protein signatures of unstable and stable atherosclerosis. Key proteins and pathway networks are identified such as the innate immune system, and neutrophil degranulation. The latter includes calprotectin S100A8/A9, which we validate in mouse and human unstable plaques, and we demonstrate the plaque-stabilizing effects of its inhibition. Overall, we provide critical insights into the unique proteomic landscape of unstable atherosclerosis (as distinct from stable atherosclerosis and vascular tissue). We further establish the TS model as a reliable preclinical tool for the discovery and testing of plaque-stabilizing drugs. Finally, we provide a knowledge resource defining unstable atherosclerosis that will facilitate the identification and validation of long-sought-after therapeutic targets and drugs for plaque stabilization.

Structure and proteomic analysis of the crown-of-thorns starfish (Acanthaster sp.) radial nerve cord.

The nervous system of the Asteroidea (starfish or seastar) consists of radial nerve cords (RNCs) that interconnect with a ring nerve. Despite its relative simplicity, it facilitates the movement of multiple arms and numerous tube feet, as well as regeneration of damaged limbs. Here, we investigated the RNC ultrastructure and its molecular components within the of Pacific crown-of-thorns starfish (COTS; Acanthaster sp.), a well-known coral predator that in high-density outbreaks has major ecological impacts on coral reefs. We describe the presence of an array of unique small bulbous bulbs (40-100 µm diameter) that project from the ectoneural region of the adult RNC. Each comprise large secretory-like cells and prominent cilia. In contrast, juvenile COTS and its congener Acanthaster brevispinus lack these features, both of which are non-corallivorous. Proteomic analysis of the RNC (and isolated neural bulbs) provides the first comprehensive echinoderm protein database for neural tissue, including numerous secreted proteins associated with signalling, transport and defence. The neural bulbs contained several neuropeptides (e.g., bombyxin-type, starfish myorelaxant peptide, secretogranin 7B2-like, Ap15a-like, and ApNp35) and Deleted in Malignant Brain Tumor 1-like proteins. In summary, this study provides a new insight into the novel traits of COTS, a major pest on coral reefs, and a proteomics resource that can be used to develop (bio)control strategies and understand molecular mechanisms of regeneration.

Transient increase in abundance of B lineage but not myeloid-lineage cells in anterior kidney of sockeye salmon during return migration to the natal grounds.

As anadromous fish, sockeye salmon undergo complex endocrine changes when they return to their natal grounds to spawn. This is correlated with major immunological changes that will affect their response to pathogens. In spite of these challenges, salmon need to maintain sufficiently robust immunity to survive until spawning is complete, but the nature of immune adaptations during the spawning stage remains poorly understood. Our central question is to determine if sockeye salmon stimulate their immune system during the return migration and if so, whether this is a protective response. To begin answering this question, here we characterized the nature and timing of potential changes in anterior kidney immune fingerprints between salmon collected from seven different sites along the Kenai river, including the mouth of the river and two spawning sites. Our results revealed significant changes in abundance of B lineage, but not myeloid lineage cells during the spawning journey. This included early, transient and significant increases in abundance of both IgM+ and IgT+ B cells soon after fish entered the river, followed by a transient, significant increase in abundance of IgM++ secreting cells in fish caught mid-river, and ending with a return to base levels of both cell populations in fish caught at spawning sites. Further, males appeared to have higher immune activation than females, as reflected by higher abundance of IgM++ secreting cells, higher spleen index, and higher titers of serum IgM. Although roles for these newly generated IgM++ secreting cells remain unclear at this time, the data complement our previous work which supported roles for long-lived plasma cells to protect returning salmon from pathogens at their natal grounds. We conclude that sockeye salmon are capable of inducing B cell responses during their spawning journey, with males having stronger responses compared to females. B cell activation during the return journey may provide returning adults with additional protection against pathogens not encountered as juveniles.

A Crown-of-Thorns Seastar recombinant relaxin-like gonad-stimulating peptide triggers oocyte maturation and ovulation.

The Acanthaster planci species-complex [Crown-of-Thorns Seastar (COTS)] are highly fecund echinoderms that exhibit population outbreaks on coral reef ecosystems worldwide, including the Australian Great Barrier Reef. A better understanding of the COTS molecular biology is critical towards efforts in controlling outbreaks and assisting reef recovery. In seastars, the heterodimeric relaxin-like gonad stimulating peptide (RGP) is responsible for triggering a neuroendocrine cascade that regulates resumption of oocyte meiosis prior to spawning. Our comparative RNA-seq analysis indicates a general increase in RGP gene expression in the female radial nerve cord during the reproductive season. Also, the sensory tentacles demonstrate a significantly higher expression level than radial nerve cord. A recombinant COTS RGP, generated in a yeast expression system, is highly effective in inducing oocyte germinal vesicle breakdown (GVBD), followed by ovulation from ovarian fragments. The findings of this study provide a foundation for more in-depth molecular analysis of the reproductive neuroendocrine physiology of the COTS and the RGP.

Aquaculture Breeding Enhancement: Maturation and Spawning in Sea Cucumbers Using a Recombinant Relaxin-Like Gonad-Stimulating Peptide.

Wild sea cucumber resources have been rapidly exhausted and therefore there is an urgent need to develop approaches that will help restocking. Currently, there is a lack of information regarding the genes involved in sea cucumber reproductive processes. The neurohormone relaxin-like gonad-stimulating peptide (RGP) has been identified as the active gonad-stimulating peptide in sea stars (Asteroidea), which could also be present in other echinoderm groups. In this study, a sea cucumber RGP was identified and confirmed by phylogenetic analysis. A recombinant Holothuria scabra RGP was produced in the yeast Pichia pastoris and confirmed by mass spectrometry. To assess bioactivity, four levels of purification were tested in an in vitro germinal vesicle breakdown (GVBD) bioassay. The most pure form induced 98.56 ± 1.19% GVBD in H. scabra and 89.57 ± 1.19% GVBD in Holothuria leucospilota. Cruder levels of purification still resulted in some GVBD. Upon single injection into female H. scabra, the recombinant RGP induced head waving behavior followed by spawning within 90-170 min. Spawned oocytes were fertilized successfully, larvae settled and developed into juveniles. Our results provide a key finding for the development of a break-through new artificial breeding approach in sea cucumber aquaculture.

Innate immune cell signatures in a BCWD-Resistant line of rainbow trout before and after in vivo challenge with Flavobacterium psychrophilum.

Phenotypes of myeloid-lineage cells remain poorly understood in the rainbow trout, and were the focus of this study, including effects of in vivo challenge to Flavobacterium psychrophilum (Fp), the cause of Bacterial Cold Water Disease (BCWD). A genetic line was used that is highly resistant to BCWD (R-line) as well as a susceptible control line (S-line). Using flow cytometry, we describe two Pax5-negative, myeloid-lineage populations: Population 1 consisted of small cells with high SSC and strong staining for Q4E, MPO, Pu1, EBF, and IL- 1ß, which we named "neutrophil-like" cell. Population 2 had high Q4E, but weaker MPO, Pu1, EBF, and IL-1ß staining. Five days after Fp-challenge, both genetic lines had a reduced abundance of neutrophil-like cells in anterior kidney, PBL, and spleen. Pop. 2 abundance was reduced in anterior kidney, and increased in spleen. S-line fish responded more strongly to Fp-challenge compared to R-line fish. Challenged fish with a higher abundance of neutrophil-like cells had significantly lower Fp-loads after challenge, suggesting that these cells aid in the resistance to BCWD.

Differences in Small Molecule Neurotransmitter Profiles From the Crown-of-Thorns Seastar Radial Nerve Revealed Between Sexes and Following Food-Deprivation.

Neurotransmitters serve as chemical mediators of cell communication, and are known to have important roles in regulating numerous physiological and metabolic events in eumetazoans. The Crown-of-Thorns Seastar (COTS) is an asteroid echinoderm that has been the focus of numerous ecological studies due to its negative impact on coral reefs when in large numbers. Research devoted to its neural signaling, from basic anatomy to the key small neurotransmitters, would expand our current understanding of neural-driven biological processes, such as growth and reproduction, and offers a new approach to exploring the propensity for COTS population explosions and subsequent collapse. In this study we investigated the metabolomic profiles of small molecule neurotransmitters in the COTS radial nerve cord. Multivariate analysis shows differential abundance of small molecule neurotransmitters in male and female COTS, and in food-deprived individuals with significant differences between sexes in gamma-aminobutyric acid (GABA), histamine and serotonin, and significant differences in histamine and serotonin between satiation states. Annotation established that the majority of biosynthesis enzyme genes are present in the COTS genome. The spatial distribution of GABA, histamine and serotonin in the radial nerve cord was subsequently confirmed by immunolocalization; serotonin is most prominent within the ectoneural regions, including unique neural bulbs, while GABA and histamine localize primarily within neuropil fibers. Glutamic acid, which was also found in high relative abundance and is a precursor of GABA, is known as a spawning inhibitor in seastars, and as such was tested for inhibition of ovulation ex-vivo which resulted in complete inhibition of oocyte maturation and ovulation induced by 1-Methyladenine. These findings not only advance our knowledge of echinoderm neural signaling processes but also identify potential targets for developing novel approaches for COTS biocontrol.

Quality-Improvement Effort to Reduce Hypothermia Among High-Risk Infants on a Mother-Infant Unit.

BACKGROUND AND OBJECTIVE: Neonatal hypothermia is common in low birth weight (LBW) (<2500 g) and late-preterm infants (LPIs) (34 0/7-36 6/7 weeks' gestation). It can be a contributory factor for newborn admission to a NICU, resulting in maternal-infant separation and increased resource use. Our objective was to study the efficacy of a quality-improvement bundle of hypothermia preventive measures for LPIs and/or LBW infants in a mother-infant unit. METHODS: We conducted plan-do-study-act (PDSA) cycles aimed at decreasing environmental hypothermia for LPIs and/or LBW infants in a mother-infant unit with no other indications for NICU-level care. Interventions included using warm towels after delivery, a risk identification card, an occlusive hat, delayed timing of first bath, submersion instead of sponge-bathing, and conducting all assessments under a radiant warmer during the initial hours of life. We implemented these interventions in 3 PDSA cycles and followed hypothermia rates by using statistical process control methods. RESULTS: The baseline mean monthly hypothermia rate among mother-infant unit LPIs and/or LBW infants was 29.8%. Postintervention, the rate fell to 13.3% (-16.5%; P = .002). This decrease occurred in a stepwise fashion in conjunction with the PDSA cycles. In the final, full-intervention period, the rate was 10.0% (-19.8%; P = .0003). A special-cause signal shift was observed in this final period. CONCLUSIONS: Targeted interventions can significantly reduce hypothermia in otherwise healthy LPIs and/or LBW newborns and allow them to safely remain in a mother-infant unit. If applied broadly, such preventive practices could decrease preventable hypothermia in high-risk populations.

Chemical Ecology of Chemosensation in Asteroidea: Insights Towards Management Strategies of Pest Species.

Within the Phylum Echinodermata, the class Asteroidea, commonly known as starfish and sea stars, encompasses a large number of benthos inhabiting genera and species with various feeding modalities including herbivores, carnivores, omnivores and detritivores. The Asteroidea rely on chemosensation throughout their life histories including hunting prey, avoiding or deterring predators, in the formation of spawning aggregations, synchronizing gamete release and targeting appropriate locations for larval settlement. The identities of many of the chemical stimuli that mediate these physiological and behavioural processes remain unresolved even though evidence indicates they play pivotal roles in the functionality of benthic communities. Aspects of chemosensation, as well as putative chemically-mediated behaviours and the molecular mechanisms of chemoreception, within the Asteroidea are reviewed here, with particular reference to the coral reef pest the Crown-of-Thorns starfish Acanthaster planci species complex, in the context of mitigation of population outbreaks.

The neuropeptidome of the Crown-of-Thorns Starfish, Acanthaster planci.

Outbreaks of Crown-of-Thorns Starfish (COTS; Acanthaster planci) are a major cause of destruction of coral communities on the Australian Great Barrier Reef. While factors relating to population explosions and the social interactions of COTS have been well studied, little is known about the neural mechanisms underlying COTS physiology and behaviour. One of the major classes of chemical messengers that regulate physiological and behavioural processes in animals is the neuropeptides. Here, we have analysed COTS genome and transcriptome sequence data to identify neuropeptide precursor proteins in this species. A total of 48 neuropeptide precursors were identified, including homologs of neuropeptides that are evolutionarily conserved throughout the Bilateria, and others that are novel. Proteomic mass spectrometry was employed to confirm the presence of neuropeptides in extracts of radial nerve cords. These transcriptomic and proteomic resources provide a foundation for functional studies that will enable a better understanding of COTS physiology and behaviour, and may facilitate development of novel population biocontrol methods. SIGNIFICANCE: The Crown-of-Thorns Starfish (COTS) is one of the primary factors leading to coral loss on the Great Barrier Reef, Australia. Our combined gene and proteomic findings of this study reveal the COTS neuropeptidome, including both echinoderm-like neuropeptides and novel putative neuropeptides. This represents the most comprehensive neuropeptidome for an echinoderm, contributing to the evolving knowledge of the COTS molecular neurobiology that may assist towards the development of biocontrol methods.

A Comprehensive Approach to the Management of Children and Adults with Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD), a disease characterized by inadequate neutrophil killing of microbial pathogens, affects 4 to 5 per million live births. For many decades following its description, CGD was a fatal disease in childhood. With the development of effective preventive therapies and the early recognition of infectious complications, 90% of children with CGD now survive into adulthood. The management of CGD in adults includes unique challenges and potential disease manifestations. In this article, the authors discuss the current approach to the management of CGD in both children and adults. This includes a focus on the importance of a comprehensive multidisciplinary approach in the care of CGD and its potential complications. In addition, a novel approach to improving education about CGD, and subsequently improving adherence to preventive therapies, is discussed.

Effects of acute CDP-choline treatment on resting state brain oscillations in healthy volunteers.

CDP-choline (cytidine-5'-diphosphocholine) is a phospholipid used to treat cognitive disorders, presumably repairing and maintaining brain cell membranes. Additional mechanisms may include enhanced cholinergic neurotransmission as the α7 nicotinic receptor actions of choline and increased acetylcholine synthesis accompanying CDP-choline administration may modulate brain oscillations underlying cognitive processes. This study utilizes electroencephalographic (EEG) recordings in healthy volunteers to evaluate CDP-choline induction of an oscillatory response profile associated with nicotinic stimulation. Resting state EEG was acquired in 24 male volunteers administered low (500mg) and moderate (1000mg) doses of CDP-choline in a randomized placebo-controlled, crossover trial. Consistent with nicotinic agonist treatment, spectral analysis showed dose-dependent reductions in delta and increases in alpha oscillations, which were also accompanied by decreases in beta and gamma oscillatory activity. These findings support the posit that CDP-choline cognitive enhancement involves multiple mechanisms including facilitated nicotinic cholinergic action.

Neurocognitive effects of acute choline supplementation in low, medium and high performer healthy volunteers.

Novel pharmacological treatments targeting alpha 7 nicotinic acetylcholine receptor (α7 nAChR) hypofunction in schizophrenia have shown mixed success in ameliorating cognitive impairments associated with this disorder. Choline, a selective agonist at α7 receptors is increased with oral administration of cytidine 5'-diphosphocholine (CDP-choline), the cognitive effects of which were assessed in healthy volunteers. Using the CogState test battery, behavioral performance in schizophrenia-relevant cognitive domains was assessed in 24 male participants following a single low (500mg) and moderate (1000mg) dose of CDP-choline. Relative to placebo, CDP-choline improved processing speed, working memory, verbal learning, verbal memory, and executive function in low baseline performers, while exerting no effects in medium baseline performers, and diminishing cognition in high baseline performers. Dose effects varied with cognitive domain but were evident with both the 500mg and 1000mg doses. These preliminary findings of cognitive enhancement in relatively impaired performers are consistent with the α7 receptor mechanism and support further trials with CDP-choline as a potential pro-cognitive strategy for cognitive impairment in schizophrenia.

CDP-choline: effects of the procholine supplement on sensory gating and executive function in healthy volunteers stratified for low, medium and high P50 suppression.

Diminished auditory sensory gating and associated neurocognitive deficits in schizophrenia have been linked to altered expression and function of the alpha-7 nicotinic acetycholinergic receptor (α7 nAChR), the targeting of which may have treatment potential. Choline is a selective α7 nAChR agonist and the aim of this study was to determine whether cytidine 5'-diphosphocholine (CDP-choline), or citicoline, a dietary source of choline, increases sensory gating and cognition in healthy volunteers stratified for gating level. In a randomized, placebo-controlled, double-blind design involving acute administration of low, moderate doses (500 mg, 1000 mg) of CDP-choline, 24 healthy volunteers were assessed for auditory gating as indexed by suppression of the P50 event-related potential (ERP) in a paired-stimulus (S1, S2) paradigm, and for executive function as measured by the Groton Maze Learning Task (GMLT) of the CogState Schizophrenia Battery. CDP-choline improved gating (1000 mg) and suppression of the S2 P50 response (500 mg, 1000 mg), with the effects being selective for individuals with low gating (suppression) levels. Tentative support was also shown for increased GMLT performance (500 mg) in low suppressors. These preliminary findings with CDP-choline in a healthy, schizophrenia-like surrogate sample are consistent with a α7 nAChR mechanism and support further trials with choline as a pro-cognitive strategy.

Automated outcome classification of emergency department computed tomography imaging reports.

BACKGROUND: Reliably abstracting outcomes from free-text electronic health records remains a challenge. While automated classification of free text has been a popular medical informatics topic, performance validation using real-world clinical data has been limited. The two main approaches are linguistic (natural language processing [NLP]) and statistical (machine learning). The authors have developed a hybrid system for abstracting computed tomography (CT) reports for specified outcomes. OBJECTIVES: The objective was to measure performance of a hybrid NLP and machine learning system for automated outcome classification of emergency department (ED) CT imaging reports. The hypothesis was that such a system is comparable to medical personnel doing the data abstraction. METHODS: A secondary analysis was performed on a prior diagnostic imaging study on 3,710 blunt facial trauma victims. Staff radiologists dictated CT reports as free text, which were then deidentified. A trained data abstractor manually coded the reference standard outcome of acute orbital fracture, with a random subset double-coded for reliability. The data set was randomly split evenly into training and testing sets. Training patient reports were used as input to the Medical Language Extraction and Encoding (MedLEE) NLP tool to create structured output containing standardized medical terms and modifiers for certainty and temporal status. Findings were filtered for low certainty and past/future modifiers and then combined with the manual reference standard to generate decision tree classifiers using data mining tools Waikato Environment for Knowledge Analysis (WEKA) 3.7.5 and Salford Predictive Miner 6.6. Performance of decision tree classifiers was evaluated on the testing set with or without NLP processing. RESULTS: The performance of machine learning alone was comparable to prior NLP studies (sensitivity = 0.92, specificity = 0.93, precision = 0.95, recall = 0.93, f-score = 0.94), and the combined use of NLP and machine learning showed further improvement (sensitivity = 0.93, specificity = 0.97, precision = 0.97, recall = 0.96, f-score = 0.97). This performance is similar to, or better than, that of medical personnel in previous studies. CONCLUSIONS: A hybrid NLP and machine learning automated classification system shows promise in coding free-text electronic clinical data.

Rapid (13)C Urea Breath Test to Identify Helicobacter pylori Infection in Emergency Department Patients with Upper Abdominal Pain.

INTRODUCTION: In emergency department (ED) patients with upper abdominal pain, management includes ruling out serious diseases and providing symptomatic relief. One of the major causes of upper abdominal pain is an ulcer caused by Helicobacter pylori (H. pylori), which can be treated and cured with antibiotics. We sought to estimate the prevalence of H. pylori infection in symptomatic patients using a convenience sample at a single urban academic ED and demonstrate the feasibility of ED-based testing. METHODS: We prospectively enrolled patients with a chief complaint of pain or discomfort in the upper abdomen for 1 year from February 2011 until February 2012 at a single academic urban ED. Enrolled subjects were tested for H. pylori using a rapid point of care (13)C Urea Breath Test (UBT) [Exalenz Bioscience]. We compared patient characteristics between those who tested positive versus negative for the disease. RESULTS: A total of 205 patients with upper abdominal pain were tested over 12 months, and 24% (95% confidence interval: 19% to 30%) tested positive for H. pylori. Black subjects were more likely to test positive than white subjects (28% v. 6%, P < 0.001). Other factors, such as age and sex, were not different between the 2 groups. CONCLUSION: In our ED, H. pylori infection was present in 1 in 4 patients with epigastric pain, and testing with a UBT was feasible. Further study is needed to determine the risk factors associated with infection, the prevalence of H. pylori in other EDs, the effect of the test on ED length of stay and the costeffectiveness of an ED-based test-and-treat strategy.

Measuring value for low-acuity care across settings.

Increasing healthcare costs have created an emphasis on improving value, defined as how invested time, money, and resources improve health. The role of emergency departments (EDs) within value-driven health systems is still undetermined. Often questioned is the value of an ED visit for conditions that could be reasonably treated elsewhere such as office-based, urgent, and retail clinics. This paper presents a conceptual approach to assess the value of these low-acuity visits. It adapts an existing analytic model to highlight specific factors that impact key stakeholders' (patients, insurers, and society) assessments of the value of ED-based care compared with care in alternative settings. These factors are presented in 3 equations, 1 for each stakeholder, emphasizing how tangible and intangible benefits of care weigh against direct and indirect costs and how each perspective influences value. Aligning value among groups could allow stakeholders to influence each other and could guide rational change in the delivery of acute medical care for low-acuity conditions.