Identification of sodium channel toxins from marine cone snails of the subgenera Textilia and Afonsoconus.

Voltage-gated sodium (Na) channels are transmembrane proteins that play a critical role in electrical signaling in the nervous system and other excitable tissues. µ-Conotoxins are peptide toxins from the venoms of marine cone snails (genus Conus) that block Na channels with nanomolar potency. Most species of the subgenera Textilia and Afonsoconus are difficult to acquire; therefore, their venoms have yet to be comprehensively interrogated for µ-conotoxins.

Neonatal resuscitation experience curves: simulation based mastery learning booster sessions and skill decay patterns among pediatric residents.

Background Following neonatal resuscitation program (NRP) training, decay in clinical skills can occur. Simulation-based deliberate practice (SBDP) has been shown to maintain NRP skills to a variable extent. Our study objectives were (a) to determine whether a single 30 min simulation-based intervention that incorporates SBDP and mastery learning (ML) can effectively restore skills and prevent skill decay and (b) to compare different timing options.

Development of an Assessment for Entrustable Professional Activity (EPA) 10: Emergent Patient Management.

Introduction: Medical schools in the United States are encouraged to prepare and certify the entrustment of medical students to perform 13 core entrustable professional activities (EPAs) prior to graduation. Entrustment is defined as the informed belief that the learner is qualified to autonomously perform specific patient-care activities. Core EPA-10 is the entrustment of a graduate to care for the emergent patient.

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin μO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin μO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface.

Structure and function of μ-conotoxins, peptide-based sodium channel blockers with analgesic activity.

μ-Conotoxins block voltage-gated sodium channels (VGSCs) and compete with tetrodotoxin for binding to the sodium conductance pore. Early efforts identified µ-conotoxins that preferentially blocked the skeletal muscle subtype (NaV1.4).

Interactions of disulfide-deficient selenocysteine analogs of μ-conotoxin BuIIIB with the α-subunit of the voltage-gated sodium channel subtype 1.3.

Inhibitors of the α-subunit of the voltage-gated sodium channel subtype 1.3 (NaV 1.3) are of interest as pharmacological tools for the study of neuropathic pain associated with spinal cord injury and have potential therapeutic applications.

A disulfide tether stabilizes the block of sodium channels by the conotoxin μO§-GVIIJ.

A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG).

Cyclic analogs of galanin and neuropeptide Y by hydrocarbon stapling.

Hydrocarbon stapling is an effective strategy to stabilize the helical conformation of bioactive peptides. Here we describe application of stapling to anticonvulsant neuropeptides, galanin (GAL) and neuropeptide Y (NPY), that are implicated in modulating seizures in the brain. Dicarba bridges were rationally introduced into minimized analogs of GAL and NPY resulting in increased α-helical content, in vitro metabolic stability and n-octanol/water partitioning coefficient (logD).

Distinct disulfide isomers of μ-conotoxins KIIIA and KIIIB block voltage-gated sodium channels.

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related μ-conotoxins.

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors.

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties.

Renal calculi: emergency department diagnosis and treatment.

The acute treatment of kidney stones (urolithiasis) addresses pain management and focuses on the effects of the morbidity associated with an obstructed renal system. Minimal fluid intake, resulting in decreased urine production and a high concentration of stone-forming salts, is a leading factor in renal calculi development. Radio-opaque calcareous stones account for 70% to 75% of renal calculi.

Analgesic neuropeptide W suppresses seizures in the brain revealed by rational repositioning and peptide engineering.

Anticonvulsant neuropeptides play an important role in controlling neuronal excitability that leads to pain or seizures. Based on overlapping inhibitory mechanisms, many anticonvulsant compounds have been found to exhibit both analgesic and antiepileptic activities. An analgesic neuropeptide W (NPW) targets recently deorphanized G-protein coupled receptors.

Introduction of lipidization-cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities.

The neuropeptides galanin (GAL), neuropeptide Y (NPY) or neurotensin (NT) exhibit anticonvulsant activities mediated by their respective receptors in the brain. To transform these peptides into potential neurotherapeutics, their systemic bioavailability and metabolic stability must be improved. Our recent studies with GAL analogs suggested that an introduction of lipoamino acids in the context of oligo-Lys residues (lipidization-cationization motif) significantly increases their penetration into the brain, yielding potent antiepileptic compounds.

Role-playing simulation as an educational tool for health care personnel: developing an embedded assessment framework.

Simulation- and video game-based role-playing techniques have been proven effective in changing behavior and enhancing positive decision making in a variety of professional settings, including education, the military, and health care. Although the need for developing assessment frameworks for learning outcomes has been clearly defined, there is a significant gap between the variety of existing multimedia-based instruction and technology-mediated learning systems and the number of reliable assessment algorithms. This study, based on a mixed methodology research design, aims to develop an embedded assessment algorithm, a Knowledge Assessment Module (NOTE), to capture both user interaction with the educational tool and knowledge gained from the training.

Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery.

Galanin modulates seizures in the brain through two galanin receptor subtypes, GalR1 and GalR2. To generate systemically active galanin receptor ligands that discriminate between GalR1 and GalR2, the GalR1-preferring analogue Gal-B2 (or NAX 5055) was rationally redesigned to yield GalR2-preferring analogues. Systematic truncations of the N-terminal backbone led to [N-Me,des-Sar]Gal-B2, containing N-methyltryptophan.

Developing novel antiepileptic drugs: characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models.

The endogenous neuropeptide galanin and its associated receptors galanin receptor 1 and galanin receptor 2 are highly localized in brain limbic structures and play an important role in the control of seizures in animal epilepsy models. As such, galanin receptors provide an attractive target for the development of novel anticonvulsant drugs. Our efforts to engineer galanin analogs that can penetrate the blood-brain-barrier and suppress seizures, yielded NAX 5055 (Gal-B2), a systemically-active analog that maintains low nanomolar affinity for galanin receptors and displays a potent anticonvulsant activity.

Structural requirements for a lipoamino acid in modulating the anticonvulsant activities of systemically active galanin analogues.

Introduction of lipoamino acid (LAA), Lys-palmitoyl, and cationization into a series of galanin analogues yielded systemically active anticonvulsant compounds. To study the relationship between the LAA structure and anticonvulsant activity, orthogonally protected LAAs were synthesized in which the Lys side chain was coupled to fatty acids varying in length from C(8) to C(18) or was coupled to a monodispersed polyethylene glycol, PEG(4). Galanin receptor affinity, serum stability, lipophilicity (log D), and activity in the 6 Hz mouse model of epilepsy of each of the newly synthesized analogues were determined following systemic administration.

Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities.

Galanin is an endogenous neuropeptide that modulates seizures in the brain. Because this neuropeptide does not penetrate the blood-brain barrier, we designed truncated galanin analogues in which nonessential amino acid residues were replaced by cationic and/or lipoamino acid residues. The analogues prevented seizures in the 6 Hz mouse model of epilepsy following intraperitoneal administration.

Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus.

Described herein is a general approach to identify novel compounds using the biodiversity of a megadiverse group of animals; specifically, the phylogenetic lineage of the venomous gastropods that belong to the genus Conus ("cone snails"). Cone snail biodiversity was exploited to identify three new mu-conotoxins, BuIIIA, BuIIIB and BuIIIC, encoded by the fish-hunting species Conus bullatus. BuIIIA, BuIIIB and BuIIIC are strikingly divergent in their amino acid composition compared to previous mu-conotoxins known to target the voltage-gated Na channel skeletal muscle subtype Na(v)1.

Development of an "all-hazards" hospital disaster preparedness training course utilizing multi-modality teaching.

Objectives: The objectives of the study were to develop and evaluate an "all-hazards" hospital disaster preparedness training course that utilizes a combination of classroom lectures, skills sessions, tabletop sessions, and disaster exercises to teach the principles of hospital disaster preparedness to hospital-based employees.

Methods: Participants attended a two-day, 16-hour course, entitled Hospital Disaster Life Support (HDLS). The course was designed to address seven core competencies of disaster training for healthcare workers.

Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels.

Peptide neurotoxins from cone snails continue to supply compounds with therapeutic potential. Although several analgesic conotoxins have already reached human clinical trials, a continuing need exists for the discovery and development of novel non-opioid analgesics, such as subtype-selective sodium channel blockers. Micro-conotoxin KIIIA is representative of micro-conopeptides previously characterized as inhibitors of tetrodotoxin (TTX)-resistant sodium channels in amphibian dorsal root ganglion neurons.

Conotoxins containing nonnatural backbone spacers: cladistic-based design, chemical synthesis, and improved analgesic activity.

Disulfide-rich neurotoxins from venomous animals continue to provide compounds with therapeutic potential. Minimizing neurotoxins often results in removal of disulfide bridges or critical amino acids. To address this drug-design challenge, we explored the concept of disulfide-rich scaffolds consisting of isostere polymers and peptidic pharmacophores.

Albumin is a redox-active crowding agent that promotes oxidative folding of cysteine-rich peptides.

Oxidative folding that occurs in a crowded cellular milieu is characterized by multifaceted interactions that occur among nascent polypeptides and resident components of the endoplasmic reticulum (ER) lumen. Macromolecular crowding has been considered an essential factor in the folding of polypeptides, but the excluded volume effect has not been evaluated for small, disulfide-rich peptides. In the research presented, we examined how macromolecular crowding agents, such as albumin, ovalbumin, and polysaccharides, influenced the kinetics and thermodynamics of forming disulfide bonds in four model peptides of varying molecular size from 13 residues (1.

Synthetic muO-conotoxin MrVIB blocks TTX-resistant sodium channel NaV1.8 and has a long-lasting analgesic activity.

MuO-conotoxin MrVIB is a blocker of voltage-gated sodium channels, including TTX-sensitive and -resistant subtypes. A comprehensive characterization of this peptide has been hampered by the lack of sufficient synthetic material. Here, we describe the successful chemical synthesis and oxidative folding of MrVIB that has made an investigation of the pharmacological properties and therapeutic potential of the peptide feasible.