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The discovery of new pain therapeutics targeting human nociceptive circuitry is an emerging, exciting, and rewarding field. However, current models for evaluating prospective new therapeutics [e.g., animals and two-dimensional (2D) in vitro cultures] fail to fully recapitulate the complexity of human nociceptive neuron and dorsal horn neuron biology, significantly limiting the development of novel pain therapeutics. Here, we report human spinal organoid-on-a-chip devices for modeling the biology and electrophysiology of human nociceptive neurons and dorsal horn interneurons in nociceptive circuitry. Our device can be simply made through the integration of a membrane with a three-dimensional (3D)-printed organoid holder. By combining air-liquid interface culture and spinal organoid protocols, our devices can differentiate human stem cells into human sensori-spinal-cord organoids with dorsal spinal cord interneurons and sensory neurons. By easily transferring from culture well plates to the multiple-electrode array (MEA) system, our device also allows the plug-and-play measurement of organoid activity for testing nociceptive modulators (e.g., mustard oil, capsaicin, velvet ant venom, etc.). Our organoid-on-a-chip devices are cost-efficient, scalable, easy to use, and compatible with conventional well plates, allowing the plug-and-play measurement of spinal organoid electrophysiology. By the integration of human sensory-spinal-cord organoids with our organoid-on-a-chip devices, our method may hold the promising potential to screen and validate novel therapeutics for human pain medicine discovery.
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Dispositivos Laboratorio en un Chip , Organoides , Animales , Humanos , Nocicepción , Dolor/tratamiento farmacológico , Estudios ProspectivosRESUMEN
Glutamate signalling through the N-methyl-d-aspartate receptor (NMDAR) activates the enzyme neuronal nitric oxide synthase (nNOS) to produce the signalling molecule nitric oxide (NO). We hypothesized that disruption of the protein-protein interaction between nNOS and the scaffolding protein postsynaptic density 95 kDa (PSD95) would block NMDAR-dependent NO signalling and represent a viable therapeutic route to decrease opioid reward and relapse-like behaviour without the unwanted side effects of NMDAR antagonists. We used a conditioned place preference (CPP) paradigm to evaluate the impact of two small-molecule PSD95-nNOS inhibitors, IC87201 and ZL006, on the rewarding effects of morphine. Both IC87201 and ZL006 blocked morphine-induced CPP at doses that lacked intrinsic rewarding or aversive properties. Furthermore, in vivo fast-scan cyclic voltammetry (FSCV) was used to ascertain the impact of ZL006 on morphine-induced increases in dopamine (DA) efflux in the nucleus accumbens shell (NAc shell) evoked by electrical stimulation of the medial forebrain bundle (MFB). ZL006 attenuated morphine-induced increases in DA efflux at a dose that did not have intrinsic effects on DA transmission. We also employed multiple intravenous drug self-administration approaches to examine the impact of ZL006 on the reinforcing effects of morphine. Interestingly, ZL006 did not alter acquisition or maintenance of morphine self-administration, but reduced lever pressing in a morphine relapse test after forced abstinence. Our results provide behavioural and neurochemical support for the hypothesis that inhibition of PSD95-nNOS protein-protein interactions decreases morphine reward and relapse-like behaviour, highlighting a previously unreported application for these novel therapeutics in the treatment of opioid addiction.
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Morfina , Recompensa , Animales , Homólogo 4 de la Proteína Discs Large , Morfina/farmacología , Óxido Nítrico Sintasa de Tipo I/metabolismo , Núcleo Accumbens/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/metabolismo , RecurrenciaRESUMEN
Elevated N-methyl-D-aspartate receptor activity contributes to central sensitization. Our laboratories and others recently reported that disrupting protein-protein interactions downstream of N-methyl-D-aspartate receptors suppresses pain. Specifically, disrupting binding between the enzyme neuronal nitric oxide synthase and either its upstream (postsynaptic density 95 kDa, PSD95) or downstream (e.g. nitric oxide synthase 1 adaptor protein, NOS1AP) protein partners suppressed inflammatory and/or neuropathic pain. However, the lack of a small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor has hindered efforts to validate the therapeutic utility of disrupting the neuronal nitric oxide synthase-NOS1AP interface as an analgesic strategy. We, therefore, evaluated the ability of a putative small-molecule neuronal nitric oxide synthase-NOS1AP inhibitor ZLc002 to disrupt binding between neuronal nitric oxide synthase and NOS1AP using ex vivo, in vitro, and purified recombinant systems and asked whether ZLc002 would suppress inflammatory and neuropathic pain in vivo. In vitro, ZLc002 reduced co-immunoprecipitation of full-length NOS1AP and neuronal nitric oxide synthase in cultured neurons and in HEK293T cells co-expressing full-length neuronal nitric oxide synthase and NOS1AP. However, using a cell-free biochemical binding assay, ZLc002 failed to disrupt the in vitro binding between His-neuronal nitric oxide synthase1-299 and glutathione S-transferase-NOS1AP400-506, protein sequences containing the required binding domains for this protein-protein interaction, suggesting an indirect mode of action in intact cells. ZLc002 (4-10 mg/kg i.p.) suppressed formalin-evoked inflammatory pain in rats and reduced Fos protein-like immunoreactivity in the lumbar spinal dorsal horn. ZLc002 also suppressed mechanical and cold allodynia in a mouse model of paclitaxel-induced neuropathic pain. Anti-allodynic efficacy was sustained for at least four days of once daily repeated dosing. ZLc002 also synergized with paclitaxel when administered in combination to reduce breast (4T1) or ovarian (HeyA8) tumor cell line viability but did not alter tumor cell viability without paclitaxel. Our results verify that ZLc002 disrupts neuronal nitric oxide synthase-NOS1AP interaction in intact cells and demonstrate, for the first time, that systemic administration of a putative small-molecule inhibitor of neuronal nitric oxide synthase-NOS1AP suppresses inflammatory and neuropathic pain.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Antineoplásicos/efectos adversos , Inhibidores Enzimáticos/uso terapéutico , Neuralgia/inducido químicamente , Neuralgia/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Paclitaxel/efectos adversos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Recién Nacidos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias/tratamiento farmacológico , Neuronas , Óxido Nítrico Sintasa de Tipo I/genética , Óxido Nítrico Sintasa de Tipo I/metabolismo , Umbral del Dolor/efectos de los fármacos , Ratas , Transfección , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
BACKGROUND: Although the reference human genome sequence was declared finished in 2003, some regions of the genome remain incomplete due to their complex architecture. One such region, 1q21.1-q21.2, is of increasing interest due to its relevance to human disease and evolution. Elucidation of the exact variants behind these associations has been hampered by the repetitive nature of the region and its incomplete assembly. This region also contains 238 of the 270 human DUF1220 protein domains, which are implicated in human brain evolution and neurodevelopment. Additionally, examinations of this protein domain have been challenging due to the incomplete 1q21 build. To address these problems, a single-haplotype hydatidiform mole BAC library (CHORI-17) was used to produce the first complete sequence of the 1q21.1-q21.2 region. RESULTS: We found and addressed several inaccuracies in the GRCh37sequence of the 1q21 region on large and small scales, including genomic rearrangements and inversions, and incorrect gene copy number estimates and assemblies. The DUF1220-encoding NBPF genes required the most corrections, with 3 genes removed, 2 genes reassigned to the 1p11.2 region, 8 genes requiring assembly corrections for DUF1220 domains (~91 DUF1220 domains were misassigned), and multiple instances of nucleotide changes that reassigned the domain to a different DUF1220 subtype. These corrections resulted in an overall increase in DUF1220 copy number, yielding a haploid total of 289 copies. Approximately 20 of these new DUF1220 copies were the result of a segmental duplication from 1q21.2 to 1p11.2 that included two NBPF genes. Interestingly, this duplication may have been the catalyst for the evolutionarily important human lineage-specific chromosome 1 pericentric inversion. CONCLUSIONS: Through the hydatidiform mole genome sequencing effort, the 1q21.1-q21.2 region is complete and misassemblies involving inter- and intra-region duplications have been resolved. The availability of this single haploid sequence path will aid in the investigation of many genetic diseases linked to 1q21, including several associated with DUF1220 copy number variations. Finally, the corrected sequence identified a recent segmental duplication that added 20 additional DUF1220 copies to the human genome, and may have facilitated the chromosome 1 pericentric inversion that is among the most notable human-specific genomic landmarks.
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Cromosomas Humanos Par 1 , Genoma Humano , Evolución Biológica , Proteínas Portadoras/genética , Hibridación Genómica Comparativa , Variaciones en el Número de Copia de ADN , Ligamiento Genético , Haploidia , Humanos , Estructura Terciaria de Proteína/genética , Duplicaciones Segmentarias en el GenomaRESUMEN
BACKGROUND: Chemotherapeutic treatment results in chronic pain in an estimated 30-40 percent of patients. Limited and often ineffective treatments make the need for new therapeutics an urgent one. We compared the effects of prophylactic cannabinoids as a preventative strategy for suppressing development of paclitaxel-induced nociception. The mixed CB1/CB2 agonist WIN55,212-2 was compared with the cannabilactone CB2-selective agonist AM1710, administered subcutaneously (s.c.), via osmotic mini pumps before, during, and after paclitaxel treatment. Pharmacological specificity was assessed using CB1 (AM251) and CB2 (AM630) antagonists. The impact of chronic drug infusion on transcriptional regulation of mRNA markers of astrocytes (GFAP), microglia (CD11b) and cannabinoid receptors (CB1, CB2) was assessed in lumbar spinal cords of paclitaxel and vehicle-treated rats. RESULTS: Both WIN55,212-2 and AM1710 blocked the development of paclitaxel-induced mechanical and cold allodynia; anti-allodynic efficacy persisted for approximately two to three weeks following cessation of drug delivery. WIN55,212-2 (0.1 and 0.5 mg/kg/day s.c.) suppressed the development of both paclitaxel-induced mechanical and cold allodynia. WIN55,212-2-mediated suppression of mechanical hypersensitivity was dominated by CB1 activation whereas suppression of cold allodynia was relatively insensitive to blockade by either CB1 (AM251; 3 mg/kg/day s.c.) or CB2 (AM630; 3 mg/kg/day s.c.) antagonists. AM1710 (0.032 and 3.2 mg/kg /day) suppressed development of mechanical allodynia whereas only the highest dose (3.2 mg/kg/day s.c.) suppressed cold allodynia. Anti-allodynic effects of AM1710 (3.2 mg/kg/day s.c.) were mediated by CB2. Anti-allodynic efficacy of AM1710 outlasted that produced by chronic WIN55,212-2 infusion. mRNA expression levels of the astrocytic marker GFAP was marginally increased by paclitaxel treatment whereas expression of the microglial marker CD11b was unchanged. Both WIN55,212-2 (0.5 mg/kg/day s.c.) and AM1710 (3.2 mg/kg/day s.c.) increased CB1 and CB2 mRNA expression in lumbar spinal cord of paclitaxel-treated rats in a manner blocked by AM630. CONCLUSIONS AND IMPLICATIONS: Cannabinoids block development of paclitaxel-induced neuropathy and protect against neuropathic allodynia following cessation of drug delivery. Chronic treatment with both mixed CB1/CB2 and CB2 selective cannabinoids increased mRNA expression of cannabinoid receptors (CB1, CB2) in a CB2-dependent fashion. Our results support the therapeutic potential of cannabinoids for suppressing chemotherapy-induced neuropathy in humans.
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Analgésicos/administración & dosificación , Antineoplásicos Fitogénicos/toxicidad , Cannabinoides/administración & dosificación , Neuralgia/inducido químicamente , Neuralgia/prevención & control , Paclitaxel/toxicidad , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sistemas de Liberación de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hiperalgesia/etiología , Hiperalgesia/prevención & control , Masculino , Actividad Motora/efectos de los fármacos , Neuralgia/complicaciones , Dimensión del Dolor , Umbral del Dolor/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
Childhood acute lymphoblastic leukemia (ALL) is a significant clinical problem that can be effectively treated with vincristine, a vinca alkaloid-based chemotherapeutic agent. However, nearly all children receiving vincristine treatment develop vincristine-induced peripheral neuropathy (VIPN). The impact of adolescent vincristine treatment across the lifespan remains poorly understood. We, consequently, developed an adolescent rodent model of VIPN which can be utilized to study possible long term consequences of vincristine treatment in the developing rat. We also evaluated the therapeutic efficacy of voluntary exercise and potential impact of obesity as a genetic risk factor in this model on the development and maintenance of VIPN. Out of all the dosing regimens we evaluated, the most potent VIPN was produced by fifteen consecutive daily intraperitoneal (i.p.) vincristine injections at 100 µg/kg/day, throughout the critical period of adolescence from postnatal day 35 to 49. With this treatment, vincristine-treated animals developed hypersensitivity to mechanical and cold stimulation of the plantar hind paw surface, which outlasted the period of vincristine treatment and resolved within two weeks following the cessation of vincristine injection. By contrast, impairment in grip strength gain was delayed by vincristine treatment, emerging shortly following the termination of vincristine dosing, and persisted into early adulthood without diminishing. Interestingly, voluntary wheel running exercise prevented the development of vincristine-induced hypersensitivities to mechanical and cold stimulation. However, Zucker fa/fa obese animals did not exhibit higher risk of developing VIPN compared to lean rats. Our studies identify sensory and motor impairments produced by vincristine in adolescent animals and support the therapeutic efficacy of voluntary exercise for suppressing VIPN in developing rats.
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Monitor lizards are unique among ectothermic reptiles in that they have high aerobic capacity and distinctive cardiovascular physiology resembling that of endothermic mammals. Here, we sequence the genome of the Komodo dragon Varanus komodoensis, the largest extant monitor lizard, and generate a high-resolution de novo chromosome-assigned genome assembly for V. komodoensis using a hybrid approach of long-range sequencing and single-molecule optical mapping. Comparing the genome of V. komodoensis with those of related species, we find evidence of positive selection in pathways related to energy metabolism, cardiovascular homoeostasis, and haemostasis. We also show species-specific expansions of a chemoreceptor gene family related to pheromone and kairomone sensing in V. komodoensis and other lizard lineages. Together, these evolutionary signatures of adaptation reveal the genetic underpinnings of the unique Komodo dragon sensory and cardiovascular systems, and suggest that selective pressure altered haemostasis genes to help Komodo dragons evade the anticoagulant effects of their own saliva. The Komodo dragon genome is an important resource for understanding the biology of monitor lizards and reptiles worldwide.
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Sistema Cardiovascular , Lagartos , Aclimatación , Animales , CromosomasRESUMEN
Elevated N-methyl-D-aspartate receptor (NMDAR) activity is linked to central sensitization and chronic pain. However, NMDAR antagonists display limited therapeutic potential because of their adverse side effects. Novel approaches targeting the NR2B-PSD95-nNOS complex to disrupt signaling pathways downstream of NMDARs show efficacy in preclinical pain models. Here, we evaluated the involvement of interactions between neuronal nitric oxide synthase (nNOS) and the nitric oxide synthase 1 adaptor protein (NOS1AP) in pronociceptive signaling and neuropathic pain. TAT-GESV, a peptide inhibitor of the nNOS-NOS1AP complex, disrupted the in vitro binding between nNOS and its downstream protein partner NOS1AP but not its upstream protein partner postsynaptic density 95 kDa (PSD95). Putative inactive peptides (TAT-cp4GESV and TAT-GESVΔ1) failed to do so. Only the active peptide protected primary cortical neurons from glutamate/glycine-induced excitotoxicity. TAT-GESV, administered intrathecally (i.t.), suppressed mechanical and cold allodynia induced by either the chemotherapeutic agent paclitaxel or a traumatic nerve injury induced by partial sciatic nerve ligation. TAT-GESV also blocked the paclitaxel-induced phosphorylation at Ser15 of p53, a substrate of p38 MAPK. Finally, TAT-GESV (i.t.) did not induce NMDAR-mediated motor ataxia in the rotarod test and did not alter basal nociceptive thresholds in the radiant heat tail-flick test. These observations support the hypothesis that antiallodynic efficacy of an nNOS-NOS1AP disruptor may result, at least in part, from blockade of p38 MAPK-mediated downstream effects. Our studies demonstrate, for the first time, that disrupting nNOS-NOS1AP protein-protein interactions attenuates mechanistically distinct forms of neuropathic pain without unwanted motor ataxic effects of NMDAR antagonists.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Neuralgia/tratamiento farmacológico , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Animales , Masculino , Ratones , Neuralgia/etiología , Neuralgia/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Dimensión del Dolor , Traumatismos de los Nervios Periféricos/complicaciones , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacosRESUMEN
Excessive activation of NMDA receptor (NMDAR) signaling within the spinal dorsal horn contributes to central sensitization and the induction and maintenance of pathological pain states. However, direct antagonism of NMDARs produces undesirable side effects which limit their clinical use. NMDAR activation produces central sensitization, in part, by initiating a signaling cascade that activates the enzyme neuronal nitric oxide synthase (nNOS) and generates the signaling molecule nitric oxide. NMDAR-mediated activation of nNOS requires a scaffolding protein, postsynaptic density protein 95kDa (PSD95), which tethers nNOS to NMDARs. Thus, disrupting the protein-protein interaction between PSD95 and nNOS may inhibit pro-nociceptive signaling mechanisms downstream of NMDARs and suppress central sensitization while sparing unwanted side effects associated with NMDAR antagonists. We examined the impact of small molecule PSD95-nNOS protein-protein interaction inhibitors (ZL006, IC87201) on both nociceptive behavior and formalin-evoked Fos protein expression within the lumbar spinal cord of rats. Comparisons were made with ZL007, an inactive analog of ZL006, and the NMDAR antagonist MK-801. IC87201 and ZL006, but not ZL007, suppressed phase 2 of formalin-evoked pain behavior and decreased the number of formalin-induced Fos-like immunoreactive cells in spinal dorsal horn regions associated with nociceptive processing. MK-801 suppressed Fos protein expression in both dorsal and ventral horns. MK-801 produced motor ataxia in the rotarod test whereas IC87201 and ZL006 failed to do so. ZL006 but not ZL007 suppressed paclitaxel-induced mechanical and cold allodynia in a model of chemotherapy-induced neuropathic pain. Co-immunoprecipitation experiments revealed the presence of the PSD95-nNOS complex in lumbar spinal cord of paclitaxel-treated rats, although ZL006 did not reliably disrupt the complex in all subjects. The present findings validate use of putative small molecule PSD95-nNOS protein-protein interaction inhibitors as novel analgesics and demonstrate, for the first time, that these inhibitors suppress inflammation-evoked neuronal activation at the level of the spinal dorsal horn.
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Conducta Animal/efectos de los fármacos , Homólogo 4 de la Proteína Discs Large/antagonistas & inhibidores , Maleato de Dizocilpina/farmacología , Óxido Nítrico Sintasa de Tipo I/efectos de los fármacos , Nociceptores/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Animales , Formaldehído/farmacología , Masculino , Óxido Nítrico Sintasa de Tipo I/metabolismo , Nociceptores/metabolismo , Ratas Sprague-Dawley , Médula Espinal/metabolismoRESUMEN
We present a new method, OMSV, for accurately and comprehensively identifying structural variations (SVs) from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with or without creating or destroying restriction sites. We show that OMSV has high sensitivity and specificity, with clear performance gains over the latest method. Applying OMSV to a human cell line, we identified hundreds of SVs >2 kbp, with 68 % of them missed by sequencing-based callers. Independent experimental validation confirmed the high accuracy of these SVs. The OMSV software is available at http://yiplab.cse.cuhk.edu.hk/omsv/ .
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Variación Estructural del Genoma , Genómica/métodos , Programas Informáticos , Biología Computacional/métodos , Simulación por Computador , Genoma Humano , HumanosRESUMEN
Limitations of preclinical models of human memory contribute to the pervasive view that rodent models do not adequately predict therapeutic efficacy in producing cognitive impairments or improvements in humans. We used a source-memory model (i.e., a representation of the origin of information) we developed for use in rats to evaluate possible drug-induced impairments of both spatial memory and higher order memory functions in the same task. Memory impairment represents a major barrier to use of NMDAR antagonists as pharmacotherapies. The scaffolding protein postsynaptic density 95kDa (PSD95) links NMDARs to the neuronal enzyme nitric oxide synthase (nNOS), which catalyzes production of the signaling molecule nitric oxide (NO). Therefore, interrupting PSD95-nNOS protein-protein interactions downstream of NMDARs represents a novel therapeutic strategy to interrupt NMDAR-dependent NO signaling while bypassing unwanted side effects of NMDAR antagonists. We hypothesized that the NMDAR antagonist MK-801 would impair source memory. We also hypothesized that PSD95-nNOS inhibitors (IC87201 and ZL006) would lack the profile of cognitive impairment associated with global NMDAR antagonists. IC87201 and ZL006 suppressed NMDA-stimulated formation of cGMP, a marker of NO production, in cultured hippocampal neurons. MK-801, at doses that did not impair motor function, impaired source memory under conditions in which spatial memory was spared. Thus, source memory was more vulnerable than spatial memory to impairment. By contrast, PSD95-nNOS inhibitors, IC87201 and ZL006, administered at doses that are behaviorally effective in rats, spared source memory, spatial memory, and motor function. Thus, PSD95-nNOS inhibitors are likely to exhibit favorable therapeutic ratios compared to NMDAR antagonists.
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Maleato de Dizocilpina/toxicidad , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/toxicidad , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Trastornos de la Memoria/inducido químicamente , Óxido Nítrico Sintasa de Tipo I/metabolismo , Ácidos Aminosalicílicos/farmacología , Animales , Bencilaminas/farmacología , Células Cultivadas , Clorofenoles/farmacología , GMP Cíclico/metabolismo , Modelos Animales de Enfermedad , Homólogo 4 de la Proteína Discs Large , Embrión de Mamíferos , Hipocampo/citología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , N-Metilaspartato/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Long-Evans , Triazoles/farmacologíaRESUMEN
Comprehensive whole-genome structural variation detection is challenging with current approaches. With diploid cells as DNA source and the presence of numerous repetitive elements, short-read DNA sequencing cannot be used to detect structural variation efficiently. In this report, we show that genome mapping with long, fluorescently labeled DNA molecules imaged on nanochannel arrays can be used for whole-genome structural variation detection without sequencing. While whole-genome haplotyping is not achieved, local phasing (across >150-kb regions) is routine, as molecules from the parental chromosomes are examined separately. In one experiment, we generated genome maps from a trio from the 1000 Genomes Project, compared the maps against that derived from the reference human genome, and identified structural variations that are >5 kb in size. We find that these individuals have many more structural variants than those published, including some with the potential of disrupting gene function or regulation.
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Mapeo Cromosómico , Variación Estructural del Genoma , Análisis por Micromatrices/métodos , Línea Celular , Genoma Humano , HumanosRESUMEN
Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 µM) and ZL006 (EC50: 12.88 µM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund's adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists.
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Ácidos Aminosalicílicos/farmacología , Analgésicos/farmacología , Bencilaminas/farmacología , Clorofenoles/farmacología , Triazoles/farmacología , Animales , Ataxia/inducido químicamente , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Maleato de Dizocilpina/efectos adversos , Maleato de Dizocilpina/farmacología , Relación Dosis-Respuesta a Droga , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/fisiología , Fármacos Neuroprotectores/efectos adversos , Fármacos Neuroprotectores/farmacología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , Dolor Nociceptivo/tratamiento farmacológico , Dolor Nociceptivo/fisiopatología , Ratas Sprague-DawleyRESUMEN
Atlantic salmon and rainbow trout, like other members of the subfamily Salmoninae, are gonochoristic with male heterogamety. The finding that sex-linked genetic markers varied between species suggested that the sex-determining gene differs among salmonid species, or that there is one sex-determining gene that has the capacity to move around the genome. The discovery of sdY, the sex-determining gene in rainbow trout, and its presence in many male salmonids gave support to the latter. Additional evidence for a salmonid-specific, sex-determining jumping gene came from the mapping of the sex-determining locus to three different chromosomes in Tasmanian male Atlantic salmon lineages. To characterize the sex-determining region, we isolated three sdY containing BACs from an Atlantic salmon male library. Sequencing of these BACs yielded two contigs, one of which contained the sdY gene. Sequence analysis of the borders of male-specific and female/male common regions revealed highly repetitive sequences associated with mobile elements, which may allow an sdY cassette to jump around the genome. FISH analysis using a BAC or a plasmid containing the sdY gene showed that the sdY gene did indeed localize to the chromosomes where SEX had been mapped in different Tasmanian Atlantic salmon families. Moreover, the plasmid sdY gene probe hybridized primarily to one of the sex chromosomes as would be expected of a male-specific gene. Our results suggest that a common salmonid sex-determining gene (sdY) can move between three specific loci on chromosomes 2, 3, and 6, giving the impression that there are multiple SEX loci both within and between salmonid species.
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Genes sry , Inestabilidad Genómica , Salmo salar/genética , Animales , Cromosomas/genéticaRESUMEN
NMDA receptors (NMDAR) are glutamate-gated calcium channels that play pivotal roles in fundamental aspects of neuronal function. Dysregulated receptor function contributes to many disorders. Recruitment by NMDARs of calcium-dependent enzyme nNOS via PSD95 is seen as a key contributor to neuronal dysfunction. nNOS adaptor protein (NOS1AP), originally described as a competitor of PSD95:nNOS interaction, is regarded an inhibitor of NMDAR-driven nNOS function. In conditions of NMDAR hyperactivity such as excitotoxicity, one expects NOS1AP to be neuroprotective. Conditions of NMDAR hypoactivity, as thought to occur in schizophrenia, might be exacerbated by NOS1AP. Indeed GWAS have implicated NOS1AP and nNOS in schizophrenia. Several studies now indicate NOS1AP can mediate rather than inhibit NMDAR/nNOS-dependent responses, including excitotoxic signaling. Yet the concept of NOS1AP as an inhibitor of nNOS predominates in studies of human disease genetics. Here we review the experimental evidence to evaluate this apparent controversy, consider whether the known functions of NOS1AP might defend neurons against NMDAR dysregulation and highlight specific areas for future investigation to shed light on the functions of this adaptor protein.
RESUMEN
Whole genome duplications (WGDs) are considered to have been a driving force in the generation of evolutionary diversity that is characteristic of higher eukaryotes. The ancestor of salmonids underwent two additional WGDs compared to mammals, one (3R) at the base of the teleost radiation and another (4R) in the common ancestor of extant salmonids. We have chosen the fatty acid binding protein (fabp) gene family as a model to study the fate of duplicated genes in teleosts following WGDs. As previously described for zebrafish, we identified two copies (fabp7a and fabp7b) of the brain-type fabp gene in several fish including rainbow smelt, but there was only a single transcript in northern pike, the closest relative of the salmonids, and two rather than the expected four fabp7 genes in Atlantic salmon, rainbow trout and grayling. A phylogenetic analysis revealed that a loss of the fabp7a gene occurred in the common ancestor of the northern pike and salmonids after it had diverged from the rainbow smelt, and that the 4R WGD then gave rise to the fabp7bI and fabp7bII observed in salmonids. This is supported by genetic mapping that placed the Atlantic salmon duplicated fabp7b genes on homeologous chromosomes. There was no evidence of neo-functionalization in the salmonid fabp7bI and fabp7bII genes based on dN/dS ratios and an examination of amino acid substitutions. Atlantic salmon fabp7bI and fabp7bII genes are both expressed broadly like fabp7b expression in northern pike. However, only Atlantic salmon fabp7bII, like its counterpart in northern pike and zebrafish, was expressed in the liver. A comparison of ~2000bp upstream of Atlantic salmon fabp7b gene duplicates revealed an insertion of 62bp in fabp7bI relative to fabp7bII. The presence of predicted transcription factor binding sites in this insertion sequence may explain the differential expression of the fabp7b gene duplicates in Atlantic salmon liver.
Asunto(s)
Encéfalo/metabolismo , Proteínas de Unión a Ácidos Grasos/genética , Duplicación de Gen , Genoma , Secuencia de Aminoácidos , Animales , Proteínas de Unión a Ácidos Grasos/química , Expresión Génica , Funciones de Verosimilitud , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Secuencias Reguladoras de Ácidos Nucleicos , Salmón , Homología de Secuencia de AminoácidoRESUMEN
Gene and genome duplications are considered to be driving forces of evolution. The relatively recent genome duplication in the common ancestor of salmonids makes this group of fish an excellent system for studying the re-diploidization process and the fates of duplicate genes. We characterized the structure and genome organization of the intestinal fatty acid binding protein (fabp2) genes in Atlantic salmon as a means of understanding the evolutionary fates of members of this protein family in teleosts. A survey of EST databases identified three unique salmonid fabp2 transcripts (fabp2aI, fabp2aII and fabp2b) compared to one transcript in zebrafish. We screened the CHORI-214 Atlantic salmon BAC library and identified BACs containing each of the three fabp2 genes. Physical mapping, genetic mapping and fluorescence in situ hybridization of Atlantic salmon chromosomes revealed that Atlantic salmon fabp2aI, fabp2aII and fabp2b correspond to separate genetic loci that reside on different chromosomes. Comparative genomic analyses indicated that these genes are related to one another by two genome duplications and a gene loss. The first genome duplication occurred in the common ancestor of all teleosts, giving rise to fabp2a and fabp2b, and the second in the common ancestor of salmonids, producing fabp2aI, fabp2aII, fabp2bI and fabp2bII. A subsequent loss of fabp2bI or fabp2bII gave the complement of fabp2 genes seen in Atlantic salmon today. There is also evidence for independent losses of fabp2b genes in zebrafish and tetraodon. Although there is no evidence for partitioning of tissue expression of fabp2 genes (i.e., sub-functionalization) in Atlantic salmon, the pattern of amino acid substitutions in Atlantic salmon and rainbow trout fabp2aI and fabp2aII suggests that neo-functionalization is occurring.