ABSTRACT | PDF
SEMINAR
Messenger system
Atmesh Kumar
Postgraduate Trainee, Department of Psychiatry, Silchar Medical College and Hospital
Introduction Neurotransmission has an anatomical infrastructure but is fundamentally a very elegant chemical operation. An understanding of the principles of chemical neurotransmission is a fundamental requirement for grasping how psychopharmacological agents work via their actions upon key molecules involved in neurotransmission. Attempting to understand normal and abnormal behaviour is futile without an appreciation of the interplay of forces lurking beneath the surface. Deciphering the microscopic world of intraneuronal signalling pathways is critical to the understanding of how neurons process information. In brief intraneuronal signalling pathways make up a microscopic nervous system within each neuron that determines its sensitivity and responsiveness to its environment in light of current circumstances and past experiences.
Cells send out messengers in the form of hormones and neurotransmitters. Receptors provide both sensitivity and selectivity in this system. Detailed knowledge the neurotransmitter receptors in the brain is crucial to developing specific therapeutic approaches to correcting unwanted nervous system activity. The known or suspected neurotransmitters in the brain already number several dozens. Classic neurotransmitters are serotonin, norepinephrine, dopamine, acetylcholine, glutamate and gamma aminobutyric acid (GABA). Some of the naturally occurring neurotransmitters similar to drugs are called God’s pharmacopeia: beta endorphine (brain’s own morphine), anandamide ( brain’s own marijuana). The great majority of drugs acting in the central nervous system act on the process of neurotransmission.
Many neurons utilize more than one neurotransmitter at a single synapse. Cotransmission often involves a monoamine coupled with a neuropeptide. Under some conditions one is released and under other conditions both are released, therefore the neurons output may involve a certain polypharmacy. Since the network of neurons send and receive information via a variety of neurotransmitters, it may therefore be not only rational but necessary to use multiple drugs with multiple neurotransmitter actions for patients with psychiatric disorders. This explain why drugs with multiple mechanisms or multiple drugs in combination are the rule in psychopharmacology practice rather than the exeption. The trick is to be able to do this rationally.
Neurotransmitters/hormones/drugs as messengers There are approximately 100 billion neurons in the brain and approximately 0.15 quadrillion synapses in the cortex. Neurons influence each other through chemical signals (neurotransmitters).
Classic neurotransmission begins with an electrical process by which neurons send electrical impulses from one part of the cell to another part of the same cell via their axons. Classic neurotransmission between neurons involves one neuron hurling a chemical messenger, or neurotransmitter, at the receptors of a second neuron. Neurons can also talk back (e.g. via nitric oxide, endocannabinoids etc.). Some neurotransmission does not need synapse at all (volume transmission).
Neurotransmitters are chemicals secreted by neurons that diffuse across a small gap to the target cell. Neurons use electrical signals as well. Neurohormones are chemicals released by neurons into the blood for action at distant targets.
An electrical impulse in the first or presynaptic neuron is converted into a chemical signal at the synapse by a process known as excitation–secretion coupling. Electrical impulse ---> voltage-sensitive sodium channels (VSSCs)/voltage-sensitive chemical channels (VSCCs) ---> neurotransmitter release ---> chemical event. The process of neurotransmission is constantly transducing chemical signals into electrical signals and electrical signals into chemical signals.
Neurotransmission can also be seen as communication from the genome of the presynaptic neuron to the genome of the postsynaptic neuron and then back from the genome of the postsynaptic neuron to the genome of the presynaptic neuron. This process involves long strings of chemical messages within both presynaptic and postsynaptic neurons, called signal-transduction cascades. Signal transduction cascades triggered by chemical neurotransmission thus involve numerous molecules, starting with neurotransmitter first messenger and proceeding to second, third, fourth, and more messengers. These are somewhat akin to a molecular “pony express.”
Cycle of neurotransmitters:
1 Synthesis
2 Release from synaptic vesicles
3 Binds to receptors
4 +/- influence on post synaptic cell
5 Broken down by enzymes
6 Reuptake of transmitter
7 Formation and storage in vesicles
Drugs can affect all stages.
Neurotransmitters in brain:
Amines e.g. Serotonin, dopamine
Pituitary peptides e.g. Corticotrophin (ACTH), growth hormone (GH)
Circulating hormones e.g. Angiotensin, calcitonin, glucagon
Hypothalamic releasing hormones e.g. Corticotrophin-releasing hormone (CRH), gondotropin releasing hormone (GnRH), luteinizing hormone releasing hormone (LHRH)
Amino acids e.g. Gamma-aminobutyric acid (GABA), glycine
Gut hormones e.g. Cholecystokinin (CCK), gastrin, motilin
Opiod peptides e.g. Dynorphin, beta-endorphin, met-encephalin
Miscellaneous peptides e.g. Bombesin, bradykinin
Gases e.g. Nitric oxide (NO), carbon monoxide (CO)
Lipid neurotransmitter e.g. Anandamide
Neurokinins/tachykinins e.g. Substance P, neurokinin A/B
Purines e.g. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), adenosine
Types of receptors Receptors for the water soluble hormones/neurotransmitters are found on the surface of the target cell, on the plasma membrane. These types of receptors are coupled to various second messenger systems which mediate the action in the target cell. Receptors for the lipid soluble hormones/neurotransmitters reside in the nucleus (and sometimes the cytoplasm) of the target cell. These can diffuse through the lipid bilayer of the plasma membrane, their receptors are located on the interior of the target cell.
Receptor locations: Cytosolic or nuclear - lipophilic ligand enters cell, often activates gene, slower response; cell membrane - lipophobic ligand can't enter cell, outer surface receptor, fast response.
Steroid hormone action: Steroid hormone passes through plasma membrane. Inside target cell, the steroid hormone binds to a specific receptor prootein. Hormone-receptor complex enters the nucleus and binds to deoxynucleic acid (DNA), causing gene transcription. Protein synthesis is induced. Protein is produced.
Action of epinephrine:
1. Epinephrine is lipophobic and needs to bind to specific receptor proteins on cell surface.
2. Acting through intermediary G proteins the hormone bound receptor activates the enzyme adenenylyl cyclase which converts ATP to cyclic AMP (cAMP).
3. cAMP performs as a secondary messenger and activates protein kinase-A, an enzyme that was previously inactive.
4. Protein kinase–A phosphorylates and activates the enzyme phosphorylase which catalyses the hydrolysis of glycogen into glucose.
IP3/Ca++ second messenger system:
1. The hormone epinephrine binds to specific receptor proteins on the cell surface.
2. Acting through G proteins, the hormone-bound receptor activates the enzyme phospholipase C, which converts membrane phospholipids into inositol 1,4,5 triphosphate (IP3).
3. IP3 diffuses through the cytoplasm and binds to receptors on the endoplasmic reticulum.
4. The binding of IP3 to the receptor stimulates the endoplasmic reticulum to release calcium (Ca++) into the cytoplasm.
5. Some of the released Ca++ binds to the receptor protein called calmodulin.
6. The Ca++/calmodulin complex activates other intracellular proteins producing the horomone effects.
Four key signal transduction cascades
1. G protein-linked
2. Ion channel-linked
3. Nuclear hormone receptors
4. Receptor tyrosine kinases.
Membrane receptor classes: Ligand binding opens or closes the channel. Ligand binding to a receptor-enzyme activates an intracellular enzyme. Ligand binding to a G protein-coupled receptor opens an ion channel or alters enzyme activity. Ligand binding to integrin receptors alters the cytoskeleton.
1. G protein coupled receptors: Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. A large family of cell surface receptors have a common structural motif, seven transmembrane a-helices. Most seven-helix receptors have domains facing the extracellular side of the plasma membrane that recognize and bind signal molecules (ligands) e.g. the b-adrenergic receptor is activated by epinephrine and norepinephrine. The signal is usually passed from a seven-helix receptor to an intracellular G protein. Seven-helix receptors are thus called GPCR, or G protein-coupled receptors. Approximately 800 different GPCRs are encoded in the human genome.
G proteins are heterotrimeric, with three subunits a, b, g. A G protein that activates cAMP formation within a cell is called a stimulatory G protein, designated Gs with alpha subunit Gsa. Gs is activated e.g. by receptors for the hormones epinephrine and glucagon. The b-adrenergic receptor is the GPCR for epinephrine. In G protein linked system second messenger is a chemical. Main signal transducers activate enzymes, open ion channels, amplify adenyl cyclase-cAMP, activates synthesis.
Signal molecule binds to G protein-linked receptor, which activates the G protein. G protein turns on adenylyl cyclase, an amplifier enzyme. Adenylyl cyclase converts ATP to cAMP. cAMP activates protein kinase A. Protein kinase A phosphorylates other proteins, leading ultimately to a cellular response.
2. Ion channel-linked receptors: Ion channels are present in virtually every living cell. In ion channel linked system second messenger is an ion. They are proteins that exist within the membrane that surrounds each cell, acting as a ‘doorway’ through which ions such as potassium (K+), sodium (Na+) and calcium (Ca2+) can pass. They are mainly of two types: ligand gated ion channels and voltage gated ion channels. Channels differ with respect to the ions they allow through, and to the way they regulate the flow of these ions. Ions cannot penetrate the lipid bilayer of cell membranes. They are transported across cell membranes with the help of ion channels. Only after Erwin Neher and Bert Sakmann developed the “patch clamp technique” was the existence of ion channels proven.
Role of ion channels: Electrical impulse generation and conduction along nerves in the central and peripheral nervous system, the heart and other organs; fluid balance within cells and across cell membranes; signal transduction within and among cells.
Characteristics: Selectivity of the channel for a particular ion, gating properties, molecular structure.
Selectivity: Ion channels are either cation or anion selective. Cations are Na+, Ca2+ or K+ and anion is Cl-. Membranes in a resting cell are relatively permeable to K+ but impermeable to Na+ and Ca2+. Drugs that open potassium channels reduce membrane excitability.
Gating: Voltage gated channels, ligand gated channels, calcium release channels, store operated calcium channels.
3. Nuclear hormone receptor structure
DNA binding domain (DBD) - Zinc finger interaction, often repeated, several configurations distinguish receptor subclasses.
Ligand binding domain (LBD) - Large, three sided cavity, envelopes ligand, largely hydrophobic amino acids lining the cavity.
Activation factor 1 (AF-1) is the site for activation by several kinase or phosphorylation pathways.
Activation factor 2 (AF-2) is essential for transcription, interaction with other protein families – one represses and one activates.
Most hydrophobic steroids are bound to plasma protein carriers. Only unbound hormones can diffuse into the target cell. Steroid hormone receptors are in the cytoplasm or nucleus. The receptor-hormone complex binds to DNA and activates or represses one or more genes. Activated genes create new messenger ribonucleic acid (mRNA) that moves back to the cytoplasm. Translation produces new proteins for cell processes. Some steroid hormones also bind to membrane receptors that use second messenger systems to create rapid cellular responses.
4. Receptor tyrosine kinases: Four common structural features shared among receptor tyrosine kinases (RTKs) - extracellular ligand-binding domain, single transmembrane domain, cytoplasmic tyrosine kinase domain(s), regulatory domains.
Brief details of messengers
Examples of first messengers for four different signal transduction cascades
Neurotransmitters (G protein linked): dopamine, serotonin, norepinephrine, acetylcholine (muscarinic), glutamate (metabotropic), GABA (GABA-B), histamine.
Neurotransmitters (ionotropic, ion channel linked): glutamate (ionotropic), acetylcholine (nicotinic), GABA (GABA-A), serotonin (5HT3).
Hormones (nuclear hormone receptors): estrogen, other gonadal steroids, glucocorticoids, thyroid.
Neurotropic factors (receptor tyrosine kinases): brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF).
Examples of second messengers for four different signal transduction cascades
Generally lead to activation of protein kinases and thus phosphorylation cascades.
G protein linked: cAMP, IP3.
Ion channel linked: Ca2+.
Nuclear hormone receptors: Hormone-nuclear receptor complex.
Receptor tyrosine kinase: Alphabet soup - Ras/Ras-GTP, Raf (a protein kinase), MEK (MAP kinase E kinase kinase).
Adenylyl cyclase system
This system uses cAMP as the second messenger. cAMP is generated by the enzyme adenylyl cyclase (AC), which in turn is subjected to both stimulatory and inhibitory input from ligand-receptor complexes via G proteins (and other sources). As mentioned, Gs has stimulatory effects on AC and Gi has inhibitory effects. The activation of the respective G proteins is via two classes of receptors, one which acts on Gs and one which acts on Gi.
cAMP levels are also controlled by removal of the compound by the enzyme phosphodiesterase which converts the compound to AMP. The effects of cAMP in cells are manifest through the action of particular protein kinases, protein kinase A's or cAMP-dependent protein kinases (CADPK's). These enzymes exist as hetero-tetrameres with two regulatory and two catalytic subunits. When bound together in a complex, the catalytic subunits are inactive.
The binding of cAMP to the regulatory subunit releases the catalytic subunits which are then free to phosphorylate serine and threonine residues in target proteins and alter the activity of the phosphorylated protein. The phosphorylated protein is converted back to its original state by the action of phosphoprotein phosphatases which remove the phosphate groups.
Cyclic guanosine monophosphate (cGMP)
A second messenger regulated by neurotransmitter receptor stimulation. Different from cAMP because action is not directly mediated by G protein coupling, instead elevation in ca2+ triggers increase in NO production, which in turn activates guanylyl cyclase. Key role in mediating the response of photoreceptor cells to light. Drugs blocking cGMP phosphodiesterase in smooth muscle produce vasodilatory effects.
IP3/DAG system
It utilizes G proteins in its transduction mechanism and produces two second messengers upon stimulation by hormones. The second messenger producing enzyme is phospholipase C. This enzyme hydrolyzes phosphatidyl inositol bisphosphate (PIP2) to produce inositide triphosphate (IP3) and diacyl glycerol (DAG). The enzyme is stimulated by hormones acting through Gi proteins. The PIP2 that is hydrolyzed is produced by phosphorylating the 4 and 5 OH-groups of phosphatidyl inositol using ATP as the phosphate donor.
Of the two second messengers produced, one is hydrophilic - IP3, and one is hydrophobic – DAG. The effect of DAG is to sequester protein kinase C to the membrane and stimulate its activity. The effect of IP3 is to stimulate Ca++ release from intracellular stores in the smooth endoplasmic reticulum (SER) and mitochondria. The released Ca++ has a variety of effects one of which is the stimulation of protein kinase C.
Protein kinase C is a family of ser/thr protein kinases which include molecules whose activities require stimulation by DAG and Ca++ together, or do not require second messenger stimulation. The first type is the classical protein kinase C, but the other three types have been found by predominantly molecular biological techniques, isolated and characterised.
Gated (K+) channels Its G protein modulated second messenger system, is ligand-gated K+ channel, which acts to increase potassium conductance in post synaptic neurons act via G proteins.
Convergence and divergence at the receptor/G protein level
When one considers the G protein system overall, one has to realize that there is no one-to-one relationship between a particular G protein (or G protein type) and a particular receptor (or receptor type). A G protein can be stimulated to exchange its bound nucleotide by a number of hormone-receptor complexes (convergence), and a hormone-receptor complex can interact and stimulate more than one G protein type (divergence). In addition, a particular G protein can interact with more than one membrane enzyme, and thus effect more than one signaling pathway (divergence). An example of this is the effect of Gi's on the AC and the IP3/DAG systems.
Tyrosine protein kinase systems
Perhaps the most noticeable thing about the tyrosine protein kinase (TPK) system is that there is no "second messenger." The system also uses protein kinases to transmit and amplify signals intracellularly, however the kinase is part of receptor. The binding of hormone by the receptor results in the activation of the tyrosine protein kinase activity. The kinase activity phosphorylates tyrosine residues in substrate proteins, acting in a classical protein kinase stimulatory pathway, thus altering the primary structure and activity of the target proteins. In addition the TKP activity also cross-auto-phosphorylates the receptor/TPK complex.
This autophosphorylation provides a surface for a number of proteins, cys- rich proteins, to bind to. When bound to the membrane surface by receptor/TPK complex, these proteins can catalyze a number of reactions. Among these proteins is phospholipase C, which like other phospholipase C, catalyzes the hydrolysis of PIP2 to yield IP3 and DAG. Therefore, the TPK system is capable of stimulating the second messengers and subsequent pathways of the IP3/DAG system.
A second protein which binds to TPK-P is phosphoinositide-3-kinase which catalyzes the phosphorylation, with ATP as the phosphate donor, of the 3'-OH of inositol residues in phospholipids. This generates a family of 3'-O-P inositides which act to stimulate the protein kinase B pathways.
MAP kinase cascade
Ras interacts with another protein Raf to form an active ser/thr protein kinase, MAP kinase kinase kinase (MAPKKK), associated with the membrane. The MAPKKK stimulates a protein kinase cascade, through MAPKK and MAPK, which eventually phosphorylates various transcription factors (TF's) and other proteins (Rsk) which also phosphorylate TF's. The resultant TF-P's stimulate the production of new mRNA which in turn leads to the production of new protein and an alteration of cell activity and/or function.
JAK-STAT system
The Janus Kinase/Signal Transduction Activators of Transcription system is somewhat similar to the TPK-MAPK system. The binding of ligand causes the receptors to dimerize. JAK binds to the dimerized receptors and phosphorylates them.
STAT binds to the phosphorylate receptors. And in turn is phosphorylated by JAK; this phosphorylation results in a dimerization and activation of STAT which translocates to the nucleus where it acts as a transcription factor.
Ca+2 systems
Ca+2 is a very important regulator of cell function. It is involved in the control of a large number of motile/fusion process and can act as a second messenger (IP3/DAG system). A variety of cellular proteins act to regulate intracellular Ca+2 levels to include gated Ca+2 channels and Ca+2 pumps. These include the voltage-gated Ca+2 channel in the synaptic bulb, which allows for Ca+2 entry which in turn leads to vesicle fusion and neurotransmitter release into the synaptic cleft, and the ligand-gated Ca+2 channels of the SER and mitochondria which recognize IP3. In addition there are a variety of 'external' ligand-gated channels which respond to neurotransmitters, and a special channel, the ryanodine channel, which is triggered by Ca+2. The significance of the ryanodine channel is that it allows small changes in Ca+2 concentration to be magnified by release on 'large' intracellular stores. There are two main types of Ca+2 pumps. Those that utilize a Na+ gradient to move Ca+2 and those that use ATP directly.
Calmodulin - The intracellular effects of Ca+2 are manifest through a variety of Ca+2 binding proteins. Perhaps the most important Ca+2 binding protein is calmodulin. This small protein binds four Ca+2 ions in a cooperative manner and then interacts with a variety of proteins to alter their activity.
Among the proteins which calmodulin is known to regulate are adenylyl cyclase, phosphodiesterase, and a variety of Ca+2/calmodulin dependent protein kinases. Other proteins that Ca+2 is known to regulate are phosphoprotein phosphatases, phospholipase A2 and troponin C.
Prostaglandins
Phospholipase A2, one of the protein which can be activated by calmodulin, cleaves the fatty acid attached to the 2'-OH of glycerol in phospholipids. This fatty acid is usually arachadonic acid which is a 20-carbon poly-unsaturated (5-6, 8-9, 11-12, 14-15) fatty acid. Subsequent to this cleavage, arachidonic acid is cyclized and oxidized to produce prostaglandins. Prostaglandins generally act as autocrine and paracrine factors. They act on a number of second messenger systems to include the AC system and the IP3/DAG system to augment, control or otherwise modulate the activity of previously stimulated cells or those close by.
Nitric oxide (NO)
Ligand binding to receptor elevates Ca+2 levels (Ca+2 channel or IP3 mechanism) which stimulates the activity of nitric oxide synthetase (NOSase) resulting in the production of NO from arginine. NO diffuses to soluble guanylyl cyclase and stimulates its activity resulting in the production of cGMP. The increase in intracellular cGMP then acts via standard mechanisms. The interesting thing about this mechanism is that NO is lipid soluble and can cross membranes and stimulate neighboring cells. This mechanism means that NO can also have autocrine/paracrine effect rather than being a second messenger only.
MAPK (mitogen-activated protein kinase) family
It is mainly composed of three subtypes of kinases: extracellular signal-regulated kinase (ERK), JNK and p38. MAPK signal transduction is a strictly regulated cascade. Specific MAPK is activated by specific MAPKK which is activated by specific MAPKKK following certain cellular stimuli or stress. The pathway activation will lead to different biological consequences. Deregulation of this pathway has been found in many types of cancers.
Ras GTPases
1) Activation mutants found in 30% of human tumours.
2) Membrane bound: C-terminal CAAX motif is prenylated (farnesyl and geranylgeranyl modifications).
3) Human isoforms: H-, K-, and N-ras. Approximately 188-189 AA’s (21 kD).
4) G protein cycle.
Ras is up stream of ERK family of MAP kinases. Approximately 66% of malignant melanomas contain B-Raf mutations. Of these mutations 80% are a single V599E mutation in the kinase domain. Mutation increases Raf catalytic activity.
Examples of key third, fourth messengers (signal transduction proteins/phosphoproteins) for four different signal transduction cascades
G protein linked: activated phosphokinase A, DAG, activated phosphokinase C, activated phospholipase C, multiple phosphoproteins, CREB (cAMP response element binding protein).
Ion channel linked: calcium/calmodutin kinase (CaMK), calcineurin.
Nuclear hormone receptors: hormone/nuclear receptor complex binding to hormone response elements.
Receptor tyrosine kinases: ERK, ribosomal S6 kinase (RSK), MAPK, glycogen synthase kinase 3 (GSK-3).
Activation of fourth messenger phosphoprotein can change synthesis of neurotransmitters, alter neurotransmitters release, change conductance of ions, maintain chemical neurotransmission in a state of readiness or dormancy.
Additional examples of third, fourth and subsequent messengers
Protein kinases, protein phosphatases, G protein coupled receptors (beta-adrenergic, opoid), G protein alpha, beta, gamma subunits, Ras super family proteins, voltage gated Na+ channels, voltage gated Ca+ channels (alpha subunits), nuclear hormone receptors, DARPP-32 (dopamine and cAMP regulated phosphoprotein), n-methyl-d-aspartate (NMDA)-R1 receptors (NMDA subunit), GABA-A receptors, phospholipase-C, IP3 receptors, CaMK, Trk (antiapoptotic receptors), cytoskeletal proteins (MAP-2, Tau, myosin light chain), synaptic vesicle proteins (synapsins), transcription factors (CREB proteins).
The ultimate targets of signal transduction are phosphoproteins and genes. Active kinases shoot phosphate group into the fourth messenger phosphoproteins and phosphatases remove them. Balance between phosphorylation and dephosphorylation of fourth messenger kinases and phosphatases play a vital role in regulating many molecules critical to chemical neurotransmission. Calcium is able to activate both the kinases and phosphatases.
Transcription factors are activated when phosphorylated. They in turn activate RNA polymerase which starts transcription. Regulatory regions of genes has enhancer and promoter elements. Immediate early genes belong to a family called “leucine zippers.” They function as rapid responders to the neurotransmitter’s input. They start functioning within 15 minutes and last about half hour to one hour.
Examples of key products of late genes targeted by all four signal transduction cascades
Synthetic enzymes for neurotransmitters (e.g. tyrosine hydroxylase, tryptophan hydroxylase), growth factors (e.g. BDNF), cytoskeletal proteins, synaptic vesicle proteins, ion channels, receptors, intracellular signaling proteins, cellular adhesion molecules.
Examples of diverse biological responses due to long term effects of late gene products from all four signal transduction cascades
Synaptogenesis, strengthen a synapse, neurogenesis, apoptosis, neurodegeneration/atrophy, learning, memory, antidepressant response, psychotherapeutic response, endocrine response, increase the efficiency of information processing in circuits, bias circuits towards decreased efficiency of information processing especially under stress, production of a mental illness (e.g. chronic pain, panic disorder).
One third of psychotropic drugs target one of the transporters for a neurotransmitter. Another one third target receptors coupled to G protein. Molecular site of action of other third include ligand gated ion channels, voltage sensitive ion channels and various enzymes. Learning as well as experiences from the environment can indeed alter which genes are expressed and thus give rise to changes in neuronal connections. Thus human experiences, education and even psychotherapy may change the expression of genes and alter the distribution and strength of specific synaptic connections. Genes modify behaviour and behaviour modify genes.
References
1. Stahl’s Essential Psychopharmacology: Neuroscientific basis and practical applications; 3rd edition.
2. Neurotransmitters, Drugs and Brain Function; edited by R.A. Webster.
3. Kaplan and Sadock’s – Comprehensive Textbook of Psychiatry, 8th edition.
4. Internet.
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