Comparison of atmospheric radionuclide dispersion models for a risk-informed consequence-driven advanced reactor licensing framework.

Current nuclear facility emergency planning zones (EPZs) are based on outdated distance-based criteria, predating comprehensive dose and risk-informed frameworks. Recent advancements in simulation tools have permitted the development of site-specific, dose, and risk-based consequence-driven assessment frameworks. This study investigated the computation of advanced reactor (AR) EPZs using two atmospheric dispersion models: a straight-line Gaussian plume model (GPM) and a semi-Lagrangian Particle in Cell (PIC). Two case studies were conducted: (1) benchmarking the NRC SOARCA study for the Peach Bottom Nuclear Generating Station and (2) analyzing an advanced INL Heat Pipe Design A microreactor's end-of-cycle inventory. The dose criteria for both cases were 10 mSv at mean weather conditions and 50 mSv at 95th percentile weather conditions at 96 h post-release. Results demonstrated that GPM and PIC estimated similar mean peak dose levels for large boiling water reactors in the farfield case, placing EPZ limits beyond current regulations. For ARs with source terms remaining in the nearfield, PIC modeling without specific nearfield considerations could result in excessively high doses and inaccurate EPZ designations. PIC dispersion demonstrated an order of magnitude higher estimate of nearfield inhalation dose contribution when compared to GPM results. Both models significantly reduced EPZ sizing within the nearfield. Thus, reductions in the AR source term may eliminate the need for a separate EPZ.

Clinical, Radiographic and Histologic Evaluation of 40 Cystic Oral Lesions in 37 Cats.

Feline cystic oral lesions are uncommon and include odontogenic cysts and cystic odontogenic tumors. Accurate diagnosis requires close collaboration between the clinician's clinical and radiographic findings and the pathologist's histologic interpretations. The odontogenic cysts identified in this series include a periapical cyst, dentigerous cysts and a type of unclassified collateral cyst that appears to be a previously undefined, distinct entity in cats (UCC). Many of the cysts (52%) were unable to be classified due to insufficient diagnostic information, which often related to the associated tooth being unavailable for evaluation. Cystic odontogenic tumors included ameloblastomas, amyloid producing ameloblastomas (APA), and feline inductive odontogenic tumors (FIOT). The purpose of this case series was to assess correlations between clinical and radiographic findings, histopathologic interpretation and signalment to identify common characteristics and provide recommendations for clinicians and pathologists to optimize diagnostic efficiency and accuracy for cystic oral lesions in cats.

Total Synthesis of the Spider-Venom Peptide Hi1a.

Hi1a is a venom peptide from the Australian funnel-web spider Hadronyche infensa with a complex tertiary structure. Hi1a has neuroprotective and cardioprotective properties due to its potent inhibition of acid-sensing ion channel 1a (ASIC1a) and is currently being pursued as a novel therapy for acute ischemic events. Herein, we describe the total synthesis of Hi1a using native chemical ligation. The synthetic peptide was successfully folded and exhibited similar inhibitory activity on ASIC1a to recombinant Hi1a.

A pain-causing and paralytic ant venom glycopeptide.

Ants (Hymenoptera: Formicidae) are familiar inhabitants of most terrestrial environments. Although we are aware of the ability of many species to sting, knowledge of ant venom chemistry remains limited. Herein, we describe the discovery and characterization of an O-linked glycopeptide (Mg7a) as a major component of the venom of the ant Myrmecia gulosa. Electron transfer dissociation and higher-energy collisional dissociation tandem mass spectrometry were used to localize three α-N-acetylgalactosaminyl residues (α-GalNAc) present on the 63-residue peptide. To allow for functional studies, we synthesized the full-length glycosylated peptide via solid-phase peptide synthesis, combined with diselenide-selenoester ligation-deselenization chemistry. We show that Mg7a is paralytic and lethal to insects, and triggers pain behavior and inflammation in mammals, which it achieves through a membrane-targeting mode of action. Deglycosylation of Mg7a renders it insoluble in aqueous solution, suggesting a key solubilizing role of the O-glycans.

Order-disorder transitions of cytoplasmic N-termini in the mechanisms of P-type ATPases.

Membrane protein structure and function are modulated via interactions with their lipid environment. This is particularly true for integral membrane pumps, the P-type ATPases. These ATPases play vital roles in cell physiology, where they are associated with the transport of cations and lipids, thereby generating and maintaining crucial (electro-)chemical potential gradients across the membrane. Several pumps (Na+, K+-ATPase, H+, K+-ATPase and the plasma membrane Ca2+-ATPase) which are located in the asymmetric animal plasma membrane have been found to possess polybasic (lysine-rich) domains on their cytoplasmic surfaces, which are thought to act as phosphatidylserine (PS) binding domains. In contrast, the sarcoplasmic reticulum Ca2+-ATPase, located within an intracellular organelle membrane, does not possess such a domain. Here we focus on the lysine-rich N-termini of the plasma-membrane-bound Na+, K+- and H+, K+-ATPases. Synthetic peptides corresponding to the N-termini of these proteins were found, via quartz crystal microbalance and circular dichroism measurements, to interact via an electrostatic interaction with PS-containing membranes, thereby undergoing an increase in helical or other secondary structure content. As well as influencing ion pumping activity, it is proposed that this interaction could provide a mechanism for sensing the lipid asymmetry of the plasma membrane, which changes drastically when a cell undergoes apoptosis, i.e. programmed cell death. Thus, polybasic regions of plasma membrane-bound ion pumps could potentially perform the function of a "death sensor", signalling to a cell to reduce pumping activity and save energy.

A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene family.

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa. We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX1-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX1-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.

Native Chemical Ligation-Photodesulfurization in Flow.

Native chemical ligation (NCL) combined with desulfurization chemistry has revolutionized the way in which large polypeptides and proteins are accessed by chemical synthesis. Herein, we outline the use of flow chemistry for the ligation-based assembly of polypeptides. We also describe the development of a novel photodesulfurization transformation that, when coupled with flow NCL, enables efficient access to native polypeptides on time scales up to 2 orders of magnitude faster than current batch NCL-desulfurization methods. The power of the new ligation-photodesulfurization flow platform is showcased through the rapid synthesis of the 36 residue clinically approved HIV entry inhibitor enfuvirtide and the peptide diagnostic agent somatorelin.

Interaction of N-terminal peptide analogues of the Na+,K+-ATPase with membranes.

The Na+,K+-ATPase, which is present in the plasma membrane of all animal cells, plays a crucial role in maintaining the Na+ and K+ electrochemical potential gradients across the membrane. Recent studies have suggested that the N-terminus of the protein's catalytic α-subunit is involved in an electrostatic interaction with the surrounding membrane, which controls the protein's conformational equilibrium. However, because the N-terminus could not yet be resolved in any X-ray crystal structures, little information about this interaction is so far available. In measurements utilising poly-l-lysine as a model of the protein's lysine-rich N-terminus and using lipid vesicles of defined composition, here we have identified the most likely origin of the interaction as one between positively charged lysine residues of the N-terminus and negatively charged headgroups of phospholipids (notably phosphatidylserine) in the surrounding membrane. Furthermore, to isolate which segments of the N-terminus could be involved in membrane binding, we chemically synthesized N-terminal fragments of various lengths. Based on a combination of results from RH421 UV/visible absorbance measurements and solid-state 31P and 2H NMR using these N-terminal fragments as well as MD simulations it appears that the membrane interaction arises from lysine residues prior to the conserved LKKE motif of the N-terminus. The MD simulations indicate that the strength of the interaction varies significantly between different enzyme conformations.

Multiplexed Temporal Quantification of the Exercise-regulated Plasma Peptidome.

Exercise is extremely beneficial to whole body health reducing the risk of a number of chronic human diseases. Some of these physiological benefits appear to be mediated via the secretion of peptide/protein hormones into the blood stream. The plasma peptidome contains the entire complement of low molecular weight endogenous peptides derived from secretion, protease activity and PTMs, and is a rich source of hormones. In the current study we have quantified the effects of intense exercise on the plasma peptidome to identify novel exercise regulated secretory factors in humans. We developed an optimized 2D-LC-MS/MS method and used multiple fragmentation methods including HCD and EThcD to analyze endogenous peptides. This resulted in quantification of 5,548 unique peptides during a time course of exercise and recovery. The plasma peptidome underwent dynamic and large changes during exercise on a time-scale of minutes with many rapidly reversible following exercise cessation. Among acutely regulated peptides, many were known hormones including insulin, glucagon, ghrelin, bradykinin, cholecystokinin and secretogranins validating the method. Prediction of bioactive peptides regulated with exercise identified C-terminal peptides from Transgelins, which were increased in plasma during exercise. In vitro experiments using synthetic peptides identified a role for transgelin peptides on the regulation of cell-cycle, extracellular matrix remodeling and cell migration. We investigated the effects of exercise on the regulation of PTMs and proteolytic processing by building a site-specific network of protease/substrate activity. Collectively, our deep peptidomic analysis of plasma revealed that exercise rapidly modulates the circulation of hundreds of bioactive peptides through a network of proteases and PTMs. These findings illustrate that peptidomics is an ideal method for quantifying changes in circulating factors on a global scale in response to physiological perturbations such as exercise. This will likely be a key method for pinpointing exercise regulated factors that generate health benefits.

Visualization of the Reaction Trajectory and Transition State in a Hydrolytic Reaction Catalyzed by a Metalloenzyme.

Metallohydrolases are a vast family of enzymes that play crucial roles in numerous metabolic pathways. Several members have emerged as targets for chemotherapeutics. Knowledge about their reaction mechanisms and associated transition states greatly aids the design of potent and highly specific drug leads. By using a high-resolution crystal structure, we have probed the trajectory of the reaction catalyzed by purple acid phosphatase, an enzyme essential for the integrity of bone structure. In particular, the transition state is visualized, thus providing detailed structural information that may be exploited in the design of specific inhibitors for the development of new anti-osteoporotic chemotherapeutics.

Motor and Nonmotor Circuitry Activation Induced by Subthalamic Nucleus Deep Brain Stimulation in Patients With Parkinson Disease: Intraoperative Functional Magnetic Resonance Imaging for Deep Brain Stimulation.

OBJECTIVE: To test the hypothesis suggested by previous studies that subthalamic nucleus (STN) deep brain stimulation (DBS) in patients with Parkinson disease would affect the activity of motor and nonmotor networks, we applied intraoperative functional magnetic resonance imaging (fMRI) to patients receiving DBS. PATIENTS AND METHODS: Ten patients receiving STN DBS for Parkinson disease underwent intraoperative 1.5-T fMRI during high-frequency stimulation delivered via an external pulse generator. The study was conducted between January 1, 2013, and September 30, 2014. RESULTS: We observed blood oxygen level-dependent (BOLD) signal changes (false discovery rate <0.001) in the motor circuitry (including the primary motor, premotor, and supplementary motor cortices; thalamus; pedunculopontine nucleus; and cerebellum) and in the limbic circuitry (including the cingulate and insular cortices). Activation of the motor network was observed also after applying a Bonferroni correction (P<.001) to the data set, suggesting that across patients, BOLD changes in the motor circuitry are more consistent compared with those occurring in the nonmotor network. CONCLUSION: These findings support the modulatory role of STN DBS on the activity of motor and nonmotor networks and suggest complex mechanisms as the basis of the efficacy of this treatment modality. Furthermore, these results suggest that across patients, BOLD changes in the motor circuitry are more consistent than those in the nonmotor network. With further studies combining the use of real-time intraoperative fMRI with clinical outcomes in patients treated with DBS, functional imaging techniques have the potential not only to elucidate the mechanisms of DBS functioning but also to guide and assist in the surgical treatment of patients affected by movement and neuropsychiatric disorders. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01809613.

Structural determinants for binding to angiotensin converting enzyme 2 (ACE2) and angiotensin receptors 1 and 2.

Angiotensin converting enzyme 2 (ACE2) is a zinc carboxypeptidase involved in the renin-angiotensin system (RAS) and inactivates the potent vasopressive peptide angiotensin II (Ang II) by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single ß-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R, and angiotensin type 2 (AT2R) receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here, we further explore this region for the potential to modulate ligand specificity. In this study, (1) a library of 47 peptides derived from the C-terminal tetrapeptide sequence (-IHPF) of Ang II was synthesized and assessed for ACE2 binding, (2) the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, (3) high affinity ACE2 binding chimeric AngII analogs were then synthesized and assessed, (4) the structure of the full-length Ang II analogs were assessed by circular dichroism, and (5) the Ang II analogs were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor selectivity.

Structural basis of receptor sulfotyrosine recognition by a CC chemokine: the N-terminal region of CCR3 bound to CCL11/eotaxin-1.

Trafficking of leukocytes in immune surveillance and inflammatory responses is activated by chemokines engaging their receptors. Sulfation of tyrosine residues in peptides derived from the eosinophil chemokine receptor CCR3 dramatically enhances binding to cognate chemokines. We report the structural basis of this recognition and affinity enhancement. We describe the structure of a CC chemokine (CCL11/eotaxin-1) bound to a fragment of a chemokine receptor: residues 8­23 of CCR3, including two sulfotyrosine residues. We also show that intact CCR3 is sulfated and sulfation enhances receptor activity. The CCR3 sulfotyrosine residues form hydrophobic, salt bridge and cation-p interactions with residues that are highly conserved in CC chemokines. However, the orientation of the chemokine relative to the receptor N terminus differs substantially from those observed for two CXC chemokines, suggesting that initial binding of the receptor sulfotyrosine residues guides subsequent steps in receptor activation, thereby influencing the receptor conformational changes and signaling.

Single ß³-amino acid substitutions to MOG peptides suppress the development of experimental autoimmune encephalomyelitis.

CD4(+) T-cells play a key role in the pathogenesis of multiple sclerosis (MS). Altered peptide ligands capable of modulating T-cell autoreactivity are considered a promising strategy for development of antigen-specific therapies for MS. Since peptides are inherently unstable, the current study explored single ß-amino acid substitution as a means of stabilizing an epitope of myelin oligodendrocyte glycoprotein. ß-Amino acid substitution at position 44, the major T-cell receptor contact residue, increased the half-life of active metabolites. Vaccination with one altered peptide, MOG44ßF, conferred protection from EAE, decreased T-cell autoreactivity and pro-inflammatory cytokine production. Additional studies using MOG44ßF in an oral treatment regimen, administered after EAE induction, also attenuated disease severity. Thus, altered peptides such as those reported here may lead to the development of novel and more specific treatments for MS.

Visible light driven photoelectrochemical properties of Ti@TiO2 nanowire electrodes sensitized with core-shell Ag@Ag2S nanoparticles.

We present a model electrode system comprised of nanostructured Ti electrode sensitized with Ag@Ag2S core-shell nanoparticles (NPs) for visible light driven photoelectrochemistry studies. The nanostructured Ti electrode is coated with Ti@TiO2 nanowires (NW) to provide a high surface area for improved light absorption and efficient charge collection from the Ag@Ag2S NPs. Pronounced photoelectrochemical responses of Ag@Ag2S NPs under visible light were obtained and attributed to collective contributions of visible light sensitivity of Ag2S, the local field enhancement of Ag surface plasmon, enhanced charge collection by Ti@TiO2 NWs, and the high surface area of the nanostructured electrode system. The shell thickness and core size of the Ag@Ag2S core-shell structure can be controlled to achieve optimal photoelectrochemical performance. XPS, XRD, SEM, high resolution TEM, AC impedance, and other electrochemical methods are applied to resolve the structure-function relationship of the nanostructured Ag@Ag2S NP electrode.

Design and synthesis of truncated EGF-A peptides that restore LDL-R recycling in the presence of PCSK9 in vitro.

Disrupting the binding interaction between proprotein convertase (PCSK9) and the epidermal growth factor-like domain A (EGF-A domain) in the low-density lipoprotein receptor (LDL-R) is a promising strategy to promote LDL-R recycling and thereby lower circulating cholesterol levels. In this study, truncated 26 amino acid EGF-A analogs were designed and synthesized, and their structures were analyzed in solution and in complex with PCSK9. The most potent peptide had an increased binding affinity for PCSK9 (KD = 0.6 µM) compared with wild-type EGF-A (KD = 1.2 µM), and the ability to increase LDL-R recycling in the presence of PCSK9 in a cell-based assay.

Combined optical and electrochemical methods for studying electrochemistry at the single molecule and single particle level: recent progress and perspectives.

We present a review of recent efforts aimed at understanding interfacial charge transfer at the single molecule and single nanoparticle level using the combined methods of traditional electrochemistry and optical spectroscopy with high spatial, spectral, and temporal resolution. Elastic light scattering, surface enhanced Raman scattering (SERS), fluorescence, and electrogenerated chemiluminescence (ECL) techniques have been demonstrated to be powerful tools for the study of interfacial charge transfer events involving a single molecule or nanoparticle and for the characterization of nanostructured electrodes. It is shown that these optical methods enable the exploration of electrochemical events with improved temporal and spatial resolution which are usually obstructed by the ensemble averaging inherent in conventional electrochemical methods. In this report, the current status of the field is reviewed and challenges for future work are discussed.

Spatial and temporal variation of surface-enhanced Raman scattering at Ag nanowires in aqueous solution.

The spatial and temporal variation of local field enhanced Raman scattering (SERS) at Ag nanowires (NWs) in aqueous solution is presented for an improved understanding of the NW structure-SERS enhancement capability relationship. Crossed Ag NWs and Ag NW bundles are found to have SERS enhancement factors much higher than single Ag NWs because of the higher density of interstitials formed by strong surface plasmon coupling when the wires are close to each other. The role of the interstitials of Ag NWs is enhanced by using unpurified Ag NWs containing Ag nanoparticles or decorating the Ag NWs surface with gold nanoparticles using galvanic replacement reaction and electroless deposition methods. This leads to an improved SERS enhancement capability as compared to purified single Ag NWs. Raman imaging reveals a different temporal response of the SERS signal in aqueous solution in comparison to the photoluminescence background of Ag NWs in the absence of Raman-active molecules. Such a different temporal response can be potentially used to separate the SERS signal from the fluorescence background. The Discrete Dipole Approximation (DDA) method is used for the first time to calculate the local field intensity of two crossed and parallel Ag NWs. Heterogeneities in the SERS spatial distribution of the interstitials and their incident-light polarization dependence are illustrated by comparing the SEM image of a selected unpurified Ag NW bundle with its Raman image.

Dual transmission grating based imaging radiometer for tokamak edge and divertor plasmas.

The designs of single transmission grating based extreme ultraviolet (XUV) and vacuum ultraviolet (VUV) imaging spectrometers can be adapted to build an imaging radiometer for simultaneous measurement of both spectral ranges. This paper describes the design of such an imaging radiometer with dual transmission gratings. The radiometer will have an XUV coverage of 20-200 Å with a ∼10 Å resolution and a VUV coverage of 200-2000 Å with a ~50 Å resolution. The radiometer is designed to have a spatial view of 16°, with a 0.33° resolution and a time resolution of ~10 ms. The applications for such a radiometer include spatially resolved impurity monitoring and electron temperature measurements in the tokamak edge and the divertor. As a proof of principle, the single grating instruments were used to diagnose a low temperature reflex discharge and the relevant data is also included in this paper.

Identification of purple acid phosphatase inhibitors by fragment-based screening: promising new leads for osteoporosis therapeutics.

Purple acid phosphatases are metalloenzymes found in animals, plants and fungi. They possess a binuclear metal centre to catalyse the hydrolysis of phosphate esters and anhydrides under acidic conditions. In humans, elevated purple acid phosphatases levels in sera are correlated with the progression of osteoporosis and metabolic bone malignancies, making this enzyme a target for the development of new chemotherapeutics to treat bone-related illnesses. To date, little progress has been achieved towards the design of specific and potent inhibitors of this enzyme that have drug-like properties. Here, we have undertaken a fragment-based screening approach using a 500-compound library identifying three inhibitors of purple acid phosphatases with K(i) values in the 30-60 µm range. Ligand efficiency values are 0.39-0.44 kcal/mol per heavy atom. X-ray crystal structures of these compounds in complex with a plant purple acid phosphatases (2.3-2.7 Å resolution) have been determined and show that all bind in the active site within contact of the binuclear centre. For one of these compounds, the phenyl ring is positioned within 3.5 Å of the binuclear centre. Docking simulations indicate that the three compounds fit into the active site of human purple acid phosphatases. These studies open the way to the design of more potent and selective inhibitors of purple acid phosphatases that can be tested as anti-osteoporotic drug leads.