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The adequacy of setup margins for various sites in patients treated with helical tomotherapy was investigated. A total of 102 patients were investigated. The breakdown of the patients were as follows: Twenty-five patients each in brain, head and neck (H and N), and pelvis, while 12 patients in lung and 15 in craniospinal irradiation (CSI). Patients were immobilized on the institutional protocol. Altogether 2686 megavoltage computed tomography images were analyzed with 672, 747, 622, 333, and 312 fractions, respectively, from brain, H and N, pelvis, lung, and CSI. Overall systematic and random errors were calculated in three translational and three rotational directions. Setup margins were evaluated using van Herk formula. The calculated margins were compared with the margins in the clinical use for various directions and sites. We found that the clinical isotropic margin of 3 mm was adequate for brain patients. However, in the longitudinal direction it was found to be out of margin by 0.7 mm. In H and N, the calculated margins were well within the isotropic margin of 5 mm which is in clinical use. In pelvis, the calculated margin was within the limits, 8.3 mm versus 10 mm only in longitudinal direction, however, in vertical and lateral directions the calculated margins were out of clinical margins 11 mm versus 10 mm, and 8.7 mm versus 7.0, mm respectively. In lung, all the calculated margins were well within the margins used clinically. In CSI, the variation was found in the middle spine in the longitudinal direction. The clinical margins used in our hospital are adequate enough for sites H and N, lung, and brain, however, for CSI and pelvis the margins were found to be out of clinical margins.

The adequacy of setup margins for various sites in patients treated with helical tomotherapy was investigated. A total of 102 patients were investigated. The breakdown of the patients were as follows: Twenty-five patients each in brain, head and neck (H and N), and pelvis, while 12 patients in lung and 15 in craniospinal irradiation (CSI). Patients were immobilized on the institutional protocol. Altogether 2686 megavoltage computed tomography images were analyzed with 672, 747, 622, 333, and 312 fractions, respectively, from brain, H and N, pelvis, lung, and CSI. Overall systematic and random errors were calculated in three translational and three rotational directions. Setup margins were evaluated using van Herk formula. The calculated margins were compared with the margins in the clinical use for various directions and sites. We found that the clinical isotropic margin of 3 mm was adequate for brain patients. However, in the longitudinal direction it was found to be out of margin by 0.7 mm. In H and N, the calculated margins were well within the isotropic margin of 5 mm which is in clinical use. In pelvis, the calculated margin was within the limits, 8.3 mm versus 10 mm only in longitudinal direction, however, in vertical and lateral directions the calculated margins were out of clinical margins 11 mm versus 10 mm, and 8.7 mm versus 7.0, mm respectively. In lung, all the calculated margins were well within the margins used clinically. In CSI, the variation was found in the middle spine in the longitudinal direction. The clinical margins used in our hospital are adequate enough for sites H and N, lung, and brain, however, for CSI and pelvis the margins were found to be out of clinical margins. [ABSTRACT FROM AUTHOR]

A response from the authors of the article "Setup Error Analysis in Helical Tomotherapy Based Image-Guided Radiation Therapy Treatments," published in a 2015 issue, is presented.

Background: Concomitant with the rise in childhood obesity there has been a significant increase in the number of adolescents with clinical features of insulin resistance and prediabetes. Clinical insulin resistance and prediabetes are likely to progress to type 2 diabetes and early atherosclerosis if not targeted for early intervention. There are no efficacy trials of lifestyle intervention in this group to inform clinical practice. The primary aim of this randomised control trial (RCT) is to determine the efficacy and effectiveness of two different structured lifestyle interventions differing in diet composition on insulin sensitivity, in adolescents with clinical insulin resistance and/or prediabetes treated with metformin.Methods/design: This study protocol describes the design of an ongoing RCT. We are recruiting 108 (54 each treatment arm) 10 to 17 year olds with clinical features of insulin resistance and/or prediabetes, through physician referral, into a multi-centred RCT. All participants are prescribed metformin and participate in a diet and exercise program. The lifestyle program is the same for all participants except for diet composition. The diets are a high carbohydrate, low fat diet and a moderate carbohydrate, increased protein diet.The program commences with an intensive 3 month dietary intervention, implemented by trained dietitians, followed by a 3 month intensive gym and home based exercise program, supervised by certified physical trainers. To measure the longer term effectiveness, after the intensive intervention trial participants are managed by either their usual physician or study physician and followed up by the study dietitians for an additional 6 months. The primary outcome measure, change in insulin sensitivity, is measured at 3, 6 and 12 months.Discussion: Clinical insulin resistance and prediabetes in the paediatric population are rapidly emerging clinical problems with serious health outcomes. With appropriate management these conditions are potentially reversible or at least their progression can be delayed. This research study is the first trial designed to provide much needed data on the effective dietary management for this cohort. This study will inform clinical practice guidelines for adolescents with clinical insulin resistance and may assist in preventing metabolic complications, type 2 diabetes and early cardiovascular disease.Trial Registration: Australian and New Zealand Clinical Trials Registration Number ACTRN12608000416392. [ABSTRACT FROM AUTHOR]

Within the phylum Cnidaria, sea anemones (class Anthozoa) express a rich diversity of ion-channel peptide modulators with biomedical applications, but corollary discoveries from jellyfish (subphylum Medusozoa) are lacking. To bridge this gap, bioactivities of previously unexplored proteinaceous and small molecular weight (~15 kDa to 5 kDa) venom components were assessed in a mouse dorsal root ganglia (DRG) high-content calcium-imaging assay, known as constellation pharmacology. While the addition of crude venom led to nonspecific cell death and Fura-2 signal leakage due to pore-forming activity, purified small molecular weight fractions of venom demonstrated three main, concentration-dependent and reversible effects on defined heterogeneous cell types found in the primary cultures of mouse DRG. These three phenotypic responses are herein referred to as phenotype A, B and C: excitatory amplification (A) or inhibition (B) of KCl-induced calcium signals, and test compound-induced disturbances to baseline calcium levels (C). Most notably, certain Alatina alata venom fractions showed phenotype A effects in all DRG neurons; Physalia physalis and Chironex fleckeri fractions predominantly showed phenotype B effects in small- and medium-diameter neurons. Finally, specific Physalia physalis and Alatina alata venom components induced direct excitatory responses (phenotype C) in glial cells. These findings demonstrate a diversity of neuroactive compounds in jellyfish venom potentially targeting a constellation of ion channels and ligand-gated receptors with broad physiological implications.

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus , αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3β4, α6/α3β4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3β4 and α6/α3β4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide.

Marine tunicates produce defensive amino-acid-derived metabolites, including 2-(3,5-diiodo-4-methoxyphenyl)ethan-1-amine (DIMTA), but their mechanisms of action are rarely known. Using an assay-guided approach, we found that out of the many different sensory cells in the mouse dorsal root ganglion (DRG), DIMTA selectively affected low-threshold cold thermosensors. Whole-cell electrophysiology experiments using DRG cells, channels expressed in Xenopus oocytes, and human cell lines revealed that DIMTA blocks several potassium channels, reducing the magnitude of the afterhyperpolarization and increasing the baseline intracellular calcium concentration [Ca 2+ ] i of low-threshold cold thermosensors. When injected into mice, DIMTA increased the threshold of cold sensation by >3 °C. DIMTA may thus serve as a lead in the further design of compounds that inhibit problems in the cold-sensory system, such as cold allodynia and other neuropathic pain conditions.

Deep vein thrombosis (DVT) is a blood clot most commonly found in the leg, which can lead to fatal pulmonary embolism (PE). Compression ultrasound of the legs is the diagnostic gold standard, leading to a definitive diagnosis. However, many patients with possible symptoms are not found to have a DVT, resulting in long referral waiting times for patients and a large clinical burden for specialists. Thus, diagnosis at the point of care by non-specialists is desired. We collect images in a pre-clinical study and investigate a deep learning approach for the automatic interpretation of compression ultrasound images. Our method provides guidance for free-hand ultrasound and aids non-specialists in detecting DVT. We train a deep learning algorithm on ultrasound videos from 255 volunteers and evaluate on a sample size of 53 prospectively enrolled patients from an NHS DVT diagnostic clinic and 30 prospectively enrolled patients from a German DVT clinic. Algorithmic DVT diagnosis performance results in a sensitivity within a 95% CI range of (0.82, 0.94), specificity of (0.70, 0.82), a positive predictive value of (0.65, 0.89), and a negative predictive value of (0.99, 1.00) when compared to the clinical gold standard. To assess the potential benefits of this technology in healthcare we evaluate the entire clinical DVT decision algorithm and provide cost analysis when integrating our approach into diagnostic pathways for DVT. Our approach is estimated to generate a positive net monetary benefit at costs up to £72 to £175 per software-supported examination, assuming a willingness to pay of £20,000/QALY., (© 2021. The Author(s).)

In our efforts to discover new drugs to treat pain, we identified molleamines A-E ( 1 - 5 ) as major neuroactive components of the sea slug, Pleurobranchus forskalii , and their prey, Didemnum molle , tunicates. The chemical structures of molleamines were elucidated by spectroscopy and confirmed by the total synthesis of molleamines A ( 1 ) and C ( 3 ). Synthetic 3 completely blocked acetylcholine-induced calcium flux in peptidergic nociceptors (PNs) in the somatosensory nervous system. Compound 3 affected neither the α7 nAChR nor the muscarinic acetylcholine receptors in calcium flux assays. In addition to nociceptors, 3 partially blocked the acetylcholine-induced calcium flux in the sympathetic nervous system, including neurons from the superior cervical ganglion. Electrophysiology revealed a block of α3β4 (mouse) and α6/α3β4 (rat) nicotinic acetylcholine receptors (nAChRs), with IC 50 values of 1.4 and 3.1 μM, respectively. Molleamine C ( 3 ) is a partial antagonist, reaching a maximum block of 76-82% of the acetylcholine signal and showing no partial agonist response. Molleamine C ( 3 ) may thus provide a lead compound for the development of neuroactive compounds with unique biological properties.

In a program to identify pain treatments with low addiction potential, we isolated five steroids, conosteroids A-E ( 1 - 5 ), from the hypobranchial gland of the mollusk Conus geographus . Compounds 1 - 5 were active in a mouse dorsal root ganglion (DRG) assay that suggested that they might be analgesic. A synthetic analogue 6 was used for a detailed pharmacological study. Compound 6 significantly increased the pain threshold in mice in the hot-plate test at 2 and 50 mg/kg. Compound 6 at 500 nM antagonizes type-A γ-aminobutyric acid receptors (GABA A Rs). In a patch-clamp experiment, out of the six subunit combinations tested, 6 exhibited subtype selectivity, most strongly antagonizing α 1 β 1 γ 2 and α 4 β 3 γ 2 receptors (IC 50 1.5 and 1.0 μM, respectively). Although the structures of 1 - 6 differ from those of known neuroactive steroids, they are cell-type-selective modulators of GABA A Rs, expanding the known chemical space of neuroactive steroids.

Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from Conus episcopatus , CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested in vitro and in vivo . Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.

Current drug discovery efforts focus on identifying lead compounds acting on a molecular target associated with an established pathological state. Concerted molecular changes that occur in specific cell types during disease progression have generally not been identified. Here, we used constellation pharmacology to investigate rat dorsal root ganglion neurons using two models of peripheral nerve injury: chronic constriction injury (CCI) and spinal nerve ligation (SNL). In these well-established models of neuropathic pain, we show that the onset of chronic pain is accompanied by a dramatic, previously unreported increase in the number of bradykinin-responsive neurons, with larger increases observed after SNL relative to CCI. To define the neurons with altered expression, we charted the temporal course of molecular changes following 1, 3, 6, and 14 d after SNL injury and demonstrated that specific molecular changes have different time courses during the progression to a pain state. In particular, ATP receptors up-regulated on day 1 postinjury, whereas the increase in bradykinin receptors was gradual after day 3 postinjury. We specifically tracked changes in two subsets of neurons: peptidergic and nonpeptidergic nociceptors. Significant increases occurred in ATP responses in nAChR-expressing isolectin B4+ nonpeptidergic neurons 1 d postinjury, whereas peptidergic neurons did not display any significant change. We propose that remodeling of ion channels and receptors occurs in a concerted and cell-specific manner, resulting in the appearance of bradykinin-responsive neuronal subclasses that are relevant to chronic pain., Competing Interests: The authors declare no competing interest.

Identification and characterization of neuronal cell classes in motor circuits are essential for understanding the neural basis of behavior. It is a challenging task, especially in a non-genetic-model organism, to identify cell-specific expression of functional macromolecules. Here, we performed constellation pharmacology, calcium imaging of dissociated neurons to pharmacologically identify functional receptors expressed by vocal neurons in adult male and female African clawed frogs, Xenopus laevis . Previously we identified a population of vocal neurons called fast trill neurons (FTNs) in the amphibian parabrachial nucleus (PB) that express N -methyl-d-aspartate (NMDA) receptors and GABA and/or glycine receptors. Using constellation pharmacology, we identified four cell classes of putative fast trill neurons (pFTNs, responsive to both NMDA and GABA/glycine applications). We discovered that some pFTNs responded to the application of substance P (SP), acetylcholine (ACh), or both. Electrophysiological recordings obtained from FTNs using an ex vivo preparation verified that SP and/or ACh depolarize FTNs. Bilateral injection of ACh, SP, or their antagonists into PBs showed that ACh receptors are not sufficient but necessary for vocal production, and SP receptors play a role in shaping the morphology of vocalizations. Additionally, we discovered that the PB of adult female X. laevis also contains all the subclasses of neurons at a similar frequency as in males, despite their sexually distinct vocalizations. These results reveal novel neuromodulators that regulate X. laevis vocal production and demonstrate the power of constellation pharmacology in identifying the neuronal subtypes marked by functional expression of cell-specific receptors in non-genetic-model organisms. NEW & NOTEWORTHY Molecular profiles of neurons are critical for understanding the neuronal functions, but their identification is challenging especially in non-genetic-model organisms. Here, we characterized the functional expression of membrane macromolecules in vocal neurons of African clawed frogs, Xenopus laevis , using a technique called constellation pharmacology. We discovered that receptors for acetylcholine and/or substance P are expressed by some classes of vocal neurons, and their activation plays a role in the production of normal vocalizations.

Somatosensory neurons have historically been classified by a variety of approaches, including structural, anatomical, and genetic markers; electrophysiological properties; pharmacological sensitivities; and more recently, transcriptional profile differentiation. These methodologies, used separately, have yielded inconsistent classification schemes. Here, we describe phenotypic differences in response to pharmacological agents as measured by changes in cytosolic calcium concentration for the rapid classification of neurons in vitro; further analysis with genetic markers, whole-cell recordings, and single-cell transcriptomics validated these findings in a functional context. Using this general approach, which we refer to as tripartite constellation analysis (TCA), we focused on large-diameter dorsal-root ganglion (L-DRG) neurons with myelinated axons. Divergent responses to the K-channel antagonist, κM-conopeptide RIIIJ (RIIIJ), reliably identified six discrete functional cell classes. In two neuronal subclasses (L1 and L2), block with RIIIJ led to an increase in [Ca] i Simultaneous electrophysiology and calcium imaging showed that the RIIIJ-elicited increase in [Ca] i corresponded to different patterns of action potentials (APs), a train of APs in L1 neurons, and sporadic firing in L2 neurons. Genetically labeled mice established that L1 neurons are proprioceptors. The single-cell transcriptomes of L1 and L2 neurons showed that L2 neurons are Aδ-low-threshold mechanoreceptors. RIIIJ effects were replicated by application of the K v 1.1 selective antagonist, Dendrotoxin-K, in several L-DRG subclasses (L1, L2, L3, and L5), suggesting the presence of functional K v 1.1/K v 1.2 heteromeric channels. Using this approach on other neuronal subclasses should ultimately accelerate the comprehensive classification and characterization of individual somatosensory neuronal subclasses within a mixed population., Competing Interests: The authors declare no competing interest.

The vast complexity of native heteromeric K + channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K + channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this "zone of ignorance" in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets "asymmetric" K v channels composed of three K v 1.2 subunits and one K v 1.1 or K v 1.6 subunit with 100-fold higher apparent affinity compared with homomeric K v 1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional K v 1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative K v 1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by ( i ) covalent linkage of members of the mammalian Shaker-related K v 1 family to K v 1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K + channel-mediated currents in heterologous expression systems; ( ii ) molecular dynamics simulations of asymmetric K v 1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and ( iii ) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric K v channel complexes that operate in native tissues., Competing Interests: The authors declare no conflict of interest.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH 2 (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C 1 -C 4, C 2 -C 6 , C 3 -C 5 . The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC 50 , 10.2 μM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.

From a biological perspective, a natural product can be defined as a compound evolved by an organism for chemical interactions with another organism including prey, predator, competitor, pathogen, symbiont or host. Natural products hold tremendous potential as drug leads and have been extensively studied by chemists and biochemists in the pharmaceutical industry. However, the biological purpose for which a natural product evolved is rarely addressed. By focusing on a well-studied group of natural products-venom components from predatory marine cone snails-this review provides a rationale for why a better understanding of the evolution, biology and biochemistry of natural products will facilitate both neuroscience and the potential for drug leads. The larger goal is to establish a new sub-discipline in the broader field of neuroethology that we refer to as "Chemical Neuroethology", linking the substantial work carried out by chemists on natural products with accelerating advances in neuroethology.

This paper proposes a method for automatic classification of spectral domain OCT data for the identification of patients with retinal diseases such as Diabetic Macular Edema (DME). We address this issue as an anomaly detection problem and propose a method that not only allows the classification of the OCT volume, but also allows the identification of the individual diseased B-scans inside the volume. Our approach is based on modeling the appearance of normal OCT images with a Gaussian Mixture Model (GMM) and detecting abnormal OCT images as outliers. The classification of an OCT volume is based on the number of detected outliers. Experimental results with two different datasets show that the proposed method achieves a sensitivity and a specificity of 80% and 93% on the first dataset, and 100% and 80% on the second one. Moreover, the experiments show that the proposed method achieves better classification performance than other recently published works., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Introduction: Capecitabine is an oral prodrug of 5-fluorouracil and is commonly used oral chemotherapeutic drugs for advanced gastric and colorectal cancer. However, hand-foot syndrome (HFS) has high incidence, and once developed, the symptoms significantly impair quality of life (QOL), leading to a reduction in the dosage or discontinuation of the treatment. Effective health education should be offered to patients to promote self-identification and management on how to recognize HFS and use self-management techniques at the very beginning of chemotherapy., Materials and Methods: The present study intended to evaluate the effectiveness of structured teaching program on knowledge related to self-identification and management of HFS among patients receiving chemotherapy for colon cancer at tertiary cancer care center. Participants who fulfilled the criteria were selected using non-probability purposive sampling. The sample selected were 40 participants (20 participants in experimental group and 20 participants in control group)., Results: Among the group of 40 patients, 17 (85 %) participants in the experimental group and 17 (85 %) participants in the control group were receiving capecitabine and oxaliplatin (CAPOX) chemotherapy treatment protocol. Five (25 %) participants in the experimental group and ten (50 %) participants in the control group were receiving drug capecitabine at a dose of 2500 mg. The mean knowledge (knowledge related to self-identification and management of HFS) pretest score was 6.75 and mean knowledge posttest score was 10.25 in the experimental group which was statistically significant (p = 0.000) (p < 0.05). The mean knowledge pretest score of participants was 6.45 and mean knowledge posttest score of participants was 6.75 in the control group which was not statistically significant (p = 0.67) (p > 0.05)., Conclusion: The study showed a statistically significant improvement in knowledge scores of participants that occurred due to intervention of structured teaching program. This can be used to assess reduction in incidence of HFS in the future.

A conventional metabolic pathway leads to a specific product. In stark contrast, there are diversity-generating metabolic pathways that naturally produce different chemicals, sometimes of great diversity. We demonstrate that for one such pathway, tru, each ensuing metabolic step is slower, in parallel with the increasing potential chemical divergence generated as the pathway proceeds. Intermediates are long lived and accumulate progressively, in contrast with conventional metabolic pathways, in which the first step is rate-limiting and metabolic intermediates are short-lived. Understanding these fundamental differences enables several different practical applications, such as combinatorial biosynthesis, some of which we demonstrate here. We propose that these principles may provide a unifying framework underlying diversity-generating metabolism in many different biosynthetic pathways.

A pressing need in neurobiology is the comprehensive identification and characterization of neuronal subclasses within the mammalian nervous system. To this end, we used constellation pharmacology as a method to interrogate the neuronal and glial subclasses of the mouse cerebellum individually and simultaneously. We then evaluated the data obtained from constellation-pharmacology experiments by cluster analysis to classify cells into neuronal and glial subclasses, based on their functional expression of glutamate, acetylcholine, and GABA receptors, among other ion channels. Conantokin peptides were used to identify N-methyl-d-aspartate (NMDA) receptor subtypes, which revealed that neurons of the young mouse cerebellum expressed NR2A and NR2B NMDA receptor subunits. Additional pharmacological tools disclosed differential expression of α-amino-3-hydroxy-5-methyl-4-isoxazloepropionic, nicotinic acetylcholine, and muscarinic acetylcholine receptors in different neuronal and glial subclasses. Certain cell subclasses correlated with known attributes of granule cells, and we combined constellation pharmacology with genetically labeled neurons to identify and characterize Purkinje cells. This study illustrates the utility of applying constellation pharmacology to classify neuronal and glial subclasses in specific anatomical regions of the brain., (Copyright © 2016 the American Physiological Society.)

In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells., Biological Significance: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types., (Published by Elsevier Ltd.)

Previously we defined neuronal subclasses within the mouse peripheral nervous system using an experimental strategy called "constellation pharmacology." Here we demonstrate the broad applicability of constellation pharmacology by extending it to the CNS and specifically to the ventral respiratory column (VRC) of mouse brainstem, a region containing the neuronal network controlling respiratory rhythm. Analysis of dissociated cells from this locus revealed three major cell classes, each encompassing multiple subclasses. We broadly analyzed the combinations (constellations) of receptors and ion channels expressed within VRC cell classes and subclasses. These were strikingly different from the constellations of receptors and ion channels found in subclasses of peripheral neurons from mouse dorsal root ganglia. Within the VRC cell population, a subset of dissociated neurons responded to substance P, putatively corresponding to inspiratory pre-Bötzinger complex (preBötC) neurons. Using constellation pharmacology, we found that these substance P-responsive neurons also responded to histamine, and about half responded to bradykinin. Electrophysiological studies conducted in brainstem slices confirmed that preBötC neurons responsive to substance P exhibited similar responsiveness to bradykinin and histamine. The results demonstrate the predictive utility of constellation pharmacology for defining modulatory inputs into specific neuronal subclasses within central neuronal networks.

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). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) NaV1.2. A mutant channel of rNaV1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10(3)-fold less sensitive to GVIIJSSG than was wild-type rNaV1.2. In contrast, although rNaV1.5 was >10(4)-fold less sensitive to GVIIJSSG than NaV1.2, an rNaV1.5 mutant with a cysteine in the homologous location, rNaV1.5[L869C], was >10(3)-fold more sensitive than wild-type rNaV1.5. The susceptibility of rNaV1.2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNaVβ2 or rNaVβ4, but not that of rNaVβ1 or rNaVβ3, protected rNaV1.1 to -1.7 (excluding NaV1.5) against block by GVIIJSSG. Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which NaVβ- and NaVα-subunits are present in native neurons.

Different types of neurons diverge in function because they express their own unique set or constellation of signaling molecules, including receptors and ion channels that work in concert. We describe an approach to identify functionally divergent neurons within a large, heterogeneous neuronal population while simultaneously investigating specific isoforms of signaling molecules expressed in each. In this study we characterized two subclasses of menthol-sensitive neurons from cultures of dissociated mouse dorsal-root ganglia. Although these neurons represent a small fraction of the dorsal-root ganglia neuronal population, we were able to identify them and investigate the cell-specific constellations of ion channels and receptors functionally expressed in each subclass, using a panel of selective pharmacological tools. Differences were found in the functional expression of ATP receptors, TRPA1 channels, voltage-gated calcium-, potassium-, and sodium channels, and responses to physiologically relevant cold temperatures. Furthermore, the cell-specific responses to various stimuli could be altered through pharmacological interventions targeted to the cell-specific constellation of ion channels expressed in each menthol-sensitive subclass. In fact, the normal responses to cold temperature could be reversed in the two neuronal subclasses by the coapplication of the appropriate combination of pharmacological agents. This result suggests that the functionally integrated constellation of signaling molecules in a particular type of cell is a more appropriate target for effective pharmacological intervention than a single signaling molecule. This shift from molecular to cellular targets has important implications for basic research and drug discovery. We refer to this paradigm as "constellation pharmacology."

We describe a functional profiling strategy to identify and characterize subtypes of neurons present in a peripheral ganglion, which should be extendable to neurons in the CNS. In this study, dissociated dorsal-root ganglion neurons from mice were exposed to various pharmacological agents (challenge compounds), while at the same time the individual responses of >100 neurons were simultaneously monitored by calcium imaging. Each challenge compound elicited responses in only a subset of dorsal-root ganglion neurons. Two general types of challenge compounds were used: agonists of receptors (ionotropic and metabotropic) that alter cytoplasmic calcium concentration (receptor-agonist challenges) and compounds that affect voltage-gated ion channels (membrane-potential challenges). Notably, among the latter are K-channel antagonists, which elicited unexpectedly diverse types of calcium responses in different cells (i.e., phenotypes). We used various challenge compounds to identify several putative neuronal subtypes on the basis of their shared and/or divergent functional, phenotypic profiles. Our results indicate that multiple receptor-agonist and membrane-potential challenges may be applied to a neuronal population to identify, characterize, and discriminate among neuronal subtypes. This experimental approach can uncover constellations of plasma membrane macromolecules that are functionally coupled to confer a specific phenotypic profile on each neuronal subtype. This experimental platform has the potential to bridge a gap between systems and molecular neuroscience with a cellular-focused neuropharmacology, ultimately leading to the identification and functional characterization of all neuronal subtypes at a given locus in the nervous system.

With a focus on low-cost and low-power consumption, a miniature laser-induced fluorescence (LIF) detection system was assembled using a 635 nm red diode laser as the excitation source and a photodiode element coupled with an operational amplifier for signal collection. The primary elements of the miniature system, namely the laser and the detection system, cost a combined $70 and required only 270 mW of power for operation. When compared to conventional systems assembled using an argon-ion laser source and a photomultiplier tube, this represents a 98% decrease in the cost, and greater than 5000-fold decrease in power consumption. Quantitation of DNA on microdevices using the miniature LIF detection system was also performed with an error of less than 15%. This detection system is a step in the direction of commercializing microfluidic instrumentation by reducing the cost and power required for operation.

Artificial blood substitutes (ABS) offer an alternative to donated blood. Increasing the oxygen carrying capacity of blood plasma through the addition of a Perfluorocarbon emulsion (PFE) is one approach in creating a blood substitute. The peripheral resistance of the circulatory system may be altered depending on the rheological properties of the ABS. Measurements of the rheological behavior of mixtures of a PFE, Oxygent, and erythrocyte suspensions were conducted at room temperature at different hematocrits using sheep (nonaggregating) and swine (aggregating) erythrocytes. The pure PFE was found to be shear thinning. Adding 6 and 12 g per 100 mL of sheep blood at the various hematocrits increased the viscosity of the suspension from as low as 4% (6 g PFE/40% Hct) to as high as 26.5% (12 g PFE/plasma). The nonaggregating sheep erythrocyte and PFE mixtures exhibited Newtonian behavior. Shear thinning behavior continued upon addition of 6 and 12 g per 100 mL of swine blood at the various hematocrits, with a slight increase in viscosity in most cases. It was observed that adding 12 g of PFE (approximately 3 x intended clinical dose) to 40% Hct swine blood at room temperature led to a significant decrease in viscosity.

Nonideal mixing in many fermentation processes can lead to concentration gradients in nutrients, oxygen, and pH, among others. These gradients are likely to influence cellular behavior, growth, or yield of the fermentation process. Frequency of exposure to these gradients can be defined by the circulation time distribution (CTD). There are few examples of CTDs in the literature, and experimental determination of CTD is at best a challenging task. The goal in this study was to determine whether computational fluid dynamics (CFD) software (FLUENT 4 and MixSim) could be used to characterize the CTD in a single-impeller mixing tank. To accomplish this, CFD software was used to simulate flow fields in three different mixing tanks by meshing the tanks with a grid of elements and solving the Navier-Stokes equations using the kappa-epsilon turbulence model. Tracer particles were released from a reference zone within the simulated flow fields, particle trajectories were simulated for 30 s, and the time taken for these tracer particles to return to the reference zone was calculated. CTDs determined by experimental measurement, which showed distinct features (log-normal, bimodal, and unimodal), were compared with CTDs determined using CFD simulation. Reproducing the signal processing procedures used in each of the experiments, CFD simulations captured the characteristic features of the experimentally measured CTDs. The CFD data suggests new signal processing procedures that predict unimodal CTDs for all three tanks.

The potential for using polyelectrolyte multilayers (PEMs) to provide chromatographic functionality on continuous silica networks created from sol-gel chemistry has been evaluated by capillary electrochromatography (CEC). Construction of the PEM was achieved by flushing the column with polyelectrolytes of alternative charge, with variation of the properties of the exposed polyelectrolyte providing a unique means to vary the chromatographic surface. Variation of the exposed polyelectrolyte from poly(diallyldimethylammonium chloride) (PDDAC) to dextran sulfate (DS) allowed the direction of the electroosmotic flow (EOF) to be changed and also provided a means to vary the chromatographic capacity. Variation of negative polymer from DS to poly(styrene sulfonate) (PSS) significantly altered the EOF and the migration of peptides, with both the reversed-phase and ion-exchange capacities increasing. An alternative method for changing the column capacity was to change the thickness of the PEM, which was evaluated by anion-exchange CEC. A 70-80% increase in retention was observed for all anions without any increase in EOF suggesting significant penetration of the analytes through the PEM and interaction with buried charges within the PEM.

Silica monolithic columns suitable for implementation on microchips have been evaluated by ion-exchange capillary electrochromatography. Two different silica monoliths were created from the alkyl silane, tetramethyl orthosilicate (TMOS), by introducing a water-soluble organic polymer, poly(ethylene oxide) (PEO), with varying molecular weights into the prehydrolyzed sol. Silica monoliths created using 10 kDa PEO were found to have a much more closed gel structure with a smaller percentage of pores in the microm size range than gels created using 100 kDa PEO. Additionally, the size of the mesopores in the 100 kDa PEO monolith was 5 nm, while those in the 10 kDa PEO gel were only 3 nm. This resulted in a strong dependence of the electroosmotic flow (EOF) on the ionic strength of the background electrolyte, with substantial pore flow through the nm size pores observed in the 10 kDa PEO gel. The chromatographic performance of the monolithic columns was evaluated by ion-exchange electrochromatography, with ion-exchange sites introduced via dynamic coating with the cationic polymer, poly(diallyldimethylammonium chloride) (PDDAC). Separating a mixture of inorganic anions, the 10 kDa PEO monolithic columns showed a higher effective capacity than the 100 kDa PEO column.