RESUMEN
The N6-methyladenosine (m6A) modification of RNA is an emerging epigenetic regulatory mechanism that has been shown to participate in various pathophysiological processes. However, its involvement in modulating neuropathic pain is still poorly understood. In this study, we elucidate a functional role of the m6A demethylase alkylation repair homolog 5 (ALKBH5) in modulating trigeminal-mediated neuropathic pain. Peripheral nerve injury selectively upregulated the expression level of ALKBH5 in the injured trigeminal ganglion (TG) of rats. Blocking this upregulation in injured TGs alleviated trigeminal neuropathic pain, while mimicking the upregulation of ALKBH5 in intact TG neurons sufficiently induced pain-related behaviors. Mechanistically, histone deacetylase 11 downregulation induced by nerve injury increases histone H3 lysine 27 acetylation (H3K27ac), facilitating the binding of the transcription factor forkhead box protein D3 (FOXD3) to the Alkbh5 promoter and promoting Alkbh5 transcription. The increased ALKBH5 erases m6A sites in Htr3a messenger RNA (mRNA), resulting in an inability of YT521-B homology domain 2 (YTHDF2) to bind to Htr3a mRNA, thus causing an increase in 5-HT3A protein expression and 5-HT3 channel currents. Conversely, blocking the increased expression of ALKBH5 in the injured TG destabilizes nerve injury-induced 5-HT3A upregulation and reverses mechanical allodynia, and the effect can be blocked by 5-HT3A knockdown. Together, FOXD3-mediated transactivation of ALKBH5 promotes neuropathic pain through m6A-dependent stabilization of Htr3a mRNA in TG neurons. This mechanistic understanding may advance the discovery of new therapeutic targets for neuropathic pain management.
Asunto(s)
Neuralgia , Neuralgia del Trigémino , Animales , Ratas , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Neuralgia/genética , Neuralgia/metabolismo , ARN Mensajero/metabolismo , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional/genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Receptores de Serotonina 5-HT3/genéticaRESUMEN
microRNA (miRNA)mediated gene regulation has been studied as a therapeutic approach, but its functional regulatory mechanism in neuropathic pain is not well understood. Here, we identify that miRNA-32-5p (miR-32-5p) is a functional RNA in regulating trigeminal-mediated neuropathic pain. High-throughput sequencing and qPCR analysis showed that miR-32-5p was the most down-regulated miRNA in the injured trigeminal ganglion (TG) of rats. Intra-TG injection of miR-32-5p agomir or overexpression of miR-32-5p by lentiviral delivery in neurons of the injured TG attenuated established trigeminal neuropathic pain. miR-32-5p overexpression did not affect acute physiological pain, while miR-32-5p down-regulation in intact rats was sufficient to cause pain-related behaviors. Nerve injury increased the methylated histone occupancy of binding sites for the transcription factor glucocorticoid receptor in the miR-32-5p promoter region. Inhibition of the enzymes that catalyze H3K9me2 and H3K27me3 restored the expression of miR-32-5p and markedly attenuated pain behaviors. Further, miR-32-5ptargeted Cav3.2 T-type Ca2+ channels and decreased miR-32-5p associated with neuropathic pain caused an increase in Cav3.2 protein expression and T-type channel currents. Conversely, miR-32-5p overexpression in injured TG suppressed the increased expression of Cav3.2 and reversed mechanical allodynia. Together, we conclude that histone methylation-mediated miR-32-5p down-regulation in TG neurons regulates trigeminal neuropathic pain by targeting Cav3.2 channels.
Asunto(s)
MicroARNs , Neuralgia , Animales , Regulación hacia Abajo , Ganglios Espinales/metabolismo , Histonas/genética , Histonas/metabolismo , Metilación , MicroARNs/genética , MicroARNs/metabolismo , Neuralgia/metabolismo , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/metabolismoRESUMEN
BACKGROUND: Interleukin 24 (IL-24) has been implicated in the nociceptive signaling. However, direct evidence and the precise molecular mechanism underlying IL-24's role in peripheral nociception remain unclear. METHODS: Using patch clamp recording, molecular biological analysis, immunofluorescence labeling, siRNA-mediated knockdown approach and behavior tests, we elucidated the effects of IL-24 on sensory neuronal excitability and peripheral pain sensitivity mediated by T-type Ca2+ channels (T-type channels). RESULTS: IL-24 enhances T-type channel currents (T-currents) in trigeminal ganglion (TG) neurons in a reversible and dose-dependent manner, primarily by activating the interleukin-22 receptor 1 (IL-22R1). Furthermore, we found that the IL-24-induced T-type channel response is mediated through tyrosine-protein kinase Lyn, but not its common downstream target JAK1. IL-24 application significantly activated protein kinase A; this effect was independent of cAMP and prevented by Lyn antagonism. Inhibition of PKA prevented the IL-24-induced T-current response, whereas inhibition of protein kinase C or MAPK kinases had no effect. Functionally, IL-24 increased TG neuronal excitability and enhanced pain sensitivity to mechanical stimuli in mice, both of which were suppressed by blocking T-type channels. In a trigeminal neuropathic pain model induced by chronic constriction injury of the infraorbital nerve, inhibiting IL-22R1 signaling alleviated mechanical allodynia, which was reversed by blocking T-type channels or knocking down Cav3.2. CONCLUSION: Our findings reveal that IL-24 enhances T-currents by stimulating IL-22R1 coupled to Lyn-dependent PKA signaling, leading to TG neuronal hyperexcitability and pain hypersensitivity. Understanding the mechanism of IL-24/IL-22R1 signaling in sensory neurons may pave the way for innovative therapeutic strategies in pain management.
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Canales de Calcio Tipo T , Proteínas Quinasas Dependientes de AMP Cíclico , Receptores de Interleucina , Células Receptoras Sensoriales , Transducción de Señal , Ganglio del Trigémino , Familia-src Quinasas , Animales , Canales de Calcio Tipo T/metabolismo , Canales de Calcio Tipo T/genética , Familia-src Quinasas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Ganglio del Trigémino/metabolismo , Masculino , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Receptores de Interleucina/metabolismo , Ratones , Ratones Endogámicos C57BL , Interleucinas/metabolismoRESUMEN
Biogeochemical processes of atmospherically deposited cadmium (Cd) in soils and accumulation in rice were investigated through a three-year fully factorial atmospheric exposure experiment using Cd stable isotopes and diffusive gradients in thin films (DGT). Our results showed that approximately 37-79% of Cd in rice grains was contributed by atmospheric deposition through root and foliar uptake during the rice growing season, while the deposited Cd accounted for a small proportion of the soil pools. The highly bioavailable metals in atmospheric deposition significantly increased the soil DGT-measured bioavailable fraction; yet, this fraction rapidly aged following a first-order exponential decay model, leading to similar percentages of the bioavailable fraction in soils exposed for 1-3 years. The enrichment of light Cd isotopes in the atmospheric deposition resulted in a significant shift toward lighter Cd isotopes in rice plants. Using a modified isotopic mass balance model, foliar and root uptake of deposited Cd accounted for 47-51% and 28-36% in leaves, 41-45% and 22-30% in stems, and 45-49% and 26-30% in grains, respectively. The implications of this study are that new atmospheric deposition disproportionately contributes to the uptake of Cd in rice, and managing emissions thus becomes very important versus remediation of impacted soils.
RESUMEN
Cadmium (Cd) stable isotopes provide a novel technique to investigate the fate of Cd in the environment, but challenges exist for tracing the sources in the plants. We performed individual rice leaf and root exposures to dry and wet deposition using customized open-top chambers (OTCs) in the greenhouse and in the field next to a smelter, respectively. The field experiment also included a control without Cd deposition and a "full" treatment. The exposure experiments and isotope signatures showed that leaves can directly take up atmospheric Cd and then translocate within rice plants to other tissues, contributing 52-70% of Cd in grains, which exceeded the contribution (30-48%) by root exposure. The Cd isotopes in leaves, nodes, internodes, and grains demonstrate that roots preferentially take up Cd from wet deposition, but leaves favor uptake of Cd from dry deposition. The Cd uptake by leaves is redistributed via nodes, allowing for upward transport to the grains but preventing downward transport to the roots. Leaves favor uptake of heavy isotopes from atmospheric deposition (ΔCd114/110Leaf-Dust: 0.10 ± 0.02) but retain light isotopes and transport heavy isotopes to the nodes and further to grains. These findings highlight the contribution of atmospheric deposition to rice and Cd isotopes as a useful tracer for quantifying sources in plants when different isotopic compositions are in sources.
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Oryza , Contaminantes del Suelo , Cadmio , Hojas de la Planta/química , Isótopos/análisis , SueloRESUMEN
This study employs stable isotope analysis to investigate the mechanisms of cadmium (Cd) and zinc (Zn) interaction in the metal hyperaccumulating plant species Sedum plumbizincicola. To this end, the Cd and Zn isotope compositions of root, stem, leaf, and xylem sap samples were determined during metal uptake and translocation at different Cd and Zn concentrations. The enrichment of light isotopes of both elements in plants during uptake was less pronounced at low metal supply levels, likely reflecting the switch from a low-affinity to a high-affinity transport system at lower levels of external metal supply. The lower δ114/110Cd values of xylem sap when treated with a metabolic inhibitor decreasing the active Cd uptake further supports the preference of heavier Cd isotopes during high-affinity transport. The Δ66Znplant-initial solution or Δ66Znplant-final solution values were similar at different Cd concentrations, indicating negligible interaction of Cd in the Zn uptake process. However, decreasing Zn supply levels significantly increased the enrichment of light Cd isotopes in plants (Δ114/110Cd = -0.08) in low-Cd treatments but reduced the enrichment of light Cd isotopes in plants (Δ114/110Cd = 0.08) under high Cd conditions. A systematic enrichment of heavy Cd and light Zn isotopes was found in root-to-shoot translocation of the metals. The Cd concentrations of the growth solutions thereby had no significant impact on Zn isotope fractionation during root-to-shoot translocation. However, the Δ114/110Cdtranslocation values hint at possible competition between Cd and Zn for transporters during root-to-shoot transfer and this may impact the transport pathway of Cd. The stable isotope data demonstrate that the interactions between the two metals influenced the uptake and transport mechanisms of Cd in S. plumbizincicola but had little effect on those of Zn.
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Cadmio , Sedum , Contaminantes del Suelo , Suelo , Biodegradación Ambiental , Cadmio/análisis , Cadmio/metabolismo , Isótopos/análisis , Isótopos/metabolismo , Isótopos/farmacología , Raíces de Plantas/química , Raíces de Plantas/metabolismo , Sedum/metabolismo , Contaminantes del Suelo/análisis , Contaminantes del Suelo/metabolismo , Zinc/análisis , Isótopos de Zinc/análisis , Isótopos de Zinc/metabolismo , Isótopos de Zinc/farmacologíaRESUMEN
BACKGROUND: Adipokines, including adiponectin, are implicated in nociceptive pain; however, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using electrophysiological recording, immunostaining, molecular biological approaches and animal behaviour tests, we elucidated a pivotal role of adiponectin in regulating membrane excitability and pain sensitivity by manipulating Cav3.2 channels in trigeminal ganglion (TG) neurons. RESULTS: Adiponectin enhanced T-type Ca2+ channel currents (IT) in TG neurons through the activation of adiponectin receptor 1 (adipoR1) but independently of heterotrimeric G protein-mediated signaling. Coimmunoprecipitation revealed a physical association between AdipoR1 and casein kinase II alpha-subunits (CK2α) in the TG, and inhibiting CK2 activity by chemical inhibitor or siRNA targeting CK2α prevented the adiponectin-induced IT response. Adiponectin significantly activated protein kinase C (PKC), and this effect was abrogated by CK2α knockdown. Adiponectin increased the membrane abundance of PKC beta1 (PKCß1). Blocking PKCß1 pharmacologically or genetically abrogated the adiponectin-induced IT increase. In heterologous expression systems, activation of adipoR1 induced a selective enhancement of Cav3.2 channel currents, dependent on PKCß1 signaling. Functionally, adiponectin increased TG neuronal excitability and induced mechanical pain hypersensitivity, both attenuated by T-type channel blockade. In a trigeminal neuralgia model induced by chronic constriction injury of infraorbital nerve, blockade of adipoR1 signaling suppressed mechanical allodynia, which was prevented by silencing Cav3.2. CONCLUSION: Our study elucidates a novel signaling cascade wherein adiponectin stimulates TG Cav3.2 channels via adipoR1 coupled to a novel CK2α-dependent PKCß1. This process induces neuronal hyperexcitability and pain hypersensitivity. Insight into adipoR-Cav3.2 signaling in sensory neurons provides attractive targets for pain treatment.
Asunto(s)
Adiponectina , Canales de Calcio Tipo T , Neuronas , Nocicepción , Receptores de Adiponectina , Animales , Ratones , Adiponectina/farmacología , Dolor , Ganglio del TrigéminoRESUMEN
BACKGROUND: Trace amines, such as tyramine, are endogenous amino acid metabolites that have been hypothesized to promote headache. However, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch-clamp recording, immunostaining, molecular biological approaches and behaviour tests, we elucidated a critically functional role of tyramine in regulating membrane excitability and pain sensitivity by manipulating Kv1.4 channels in trigeminal ganglion (TG) neurons. RESULTS: Application of tyramine to TG neurons decreased the A-type K+ current (IA) in a manner dependent on trace amine-associated receptor 1 (TAAR1). Either siRNA knockdown of Gαo or chemical inhibition of ßγ subunit (Gßγ) signaling abrogated the response to tyramine. Antagonism of protein kinase C (PKC) prevented the tyramine-induced IA response, while inhibition of conventional PKC isoforms or protein kinase A elicited no such effect. Tyramine increased the membrane abundance of PKCθ in TG neurons, and either pharmacological or genetic inhibition of PKCθ blocked the TAAR1-mediated IA decrease. Furthermore, PKCθ-dependent IA suppression was mediated by Kv1.4 channels. Knockdown of Kv1.4 abrogated the TAAR1-induced IA decrease, neuronal hyperexcitability, and pain hypersensitivity. In a mouse model of migraine induced by electrical stimulation of the dura mater surrounding the superior sagittal sinus, blockade of TAAR1 signaling attenuated mechanical allodynia; this effect was occluded by lentiviral overexpression of Kv1.4 in TG neurons. CONCLUSION: These results suggest that tyramine induces Kv1.4-mediated IA suppression through stimulation of TAAR1 coupled to the Gßγ-dependent PKCθ signaling cascade, thereby enhancing TG neuronal excitability and mechanical pain sensitivity. Insight into TAAR1 signaling in sensory neurons provides attractive targets for the treatment of headache disorders such as migraine.
Asunto(s)
Nocicepción , Ganglio del Trigémino , Animales , Ratones , Neuronas , DolorRESUMEN
Soil salinity is a widespread stress in semi-arid forests worldwide, but how to manage nitrogen (N) nutrition to improve plant saline tolerance remains unclear. Here, the cuttings of a widely distributed poplar from central Asia, Populus russikki Jabl., were exposed to either normal or low nitrogen (LN) concentrations for two weeks in semi-controlled greenhouse, and then they were added with moderate salt solution or not for another two weeks to evaluate their physiological, biochemical, metabolites and transcriptomic profile changes. LN-pretreating alleviated the toxicity caused by the subsequent salt stress in the poplar plants, demonstrated by a significant reduction in the influx of Na+ and Cl- and improvement of the K+/Na+ ratio. The other salt-stressed traits were also ameliarated, indicated by the variations of chlorophyll content, PSII photochemical activity and lipid peroxidation. Stress alleviation resulted from two different processes. First, LN pretreatment caused a significant increase of non-structural carbohydrates (NSC), allowed for an increased production of osmolytes and a higher potential fueling ion transport under subsequent salt condition, along with increased transcript levels of the cation/H+ ATPase. Second, LN pretreatment enhanced the transcript levels of stress signaling components and phytohormones pathway as well as antioxidant enzyme activities. The results indicate that early restrictions of N supply could enhance posterior survival under saline stress in poplar plants, which is important for plantation programs and restoration activities in semi-arid areas.
Asunto(s)
Populus , Carbohidratos , Nitrógeno , Populus/genética , Estrés Salino , Tolerancia a la SalRESUMEN
α-Melanocyte-stimulating hormone (α-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that α-MSH suppresses the transient outward A-type K+ current (IA) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to α-MSH concentration-dependently decreased IA This α-MSH-induced IA decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated IA response. Blocking Gi/o-protein signaling with pertussis toxin or by dialysis of TG neurons with the Gßγ-blocking synthetic peptide QEHA abolished the α-MSH-mediated decrease in IA Further, α-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38α abrogated the α-MSH-induced IA response. Additionally, α-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by IA blockade. Taken together, our findings suggest that α-MSH suppresses IA by activating MC4R, which is coupled sequentially to the Gßγ complex of the Gi/o-protein and downstream class I PI3K-dependent p38α signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Dolor/metabolismo , Canales de Potasio/metabolismo , Receptor de Melanocortina Tipo 4/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/efectos de los fármacos , Ganglio del Trigémino/metabolismo , alfa-MSH/farmacología , Animales , Proteínas de Unión al GTP/metabolismo , Dolor/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 4/agonistas , Células Receptoras Sensoriales/patología , Ganglio del Trigémino/patología , Wortmanina/farmacologíaRESUMEN
BACKGROUND: Cholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown. METHODS: Using patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels. RESULTS: CCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gß antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade. CONCLUSION: Our findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gßγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.
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Proteína Tirosina Quinasa CSK/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Fosfatidilinositol 3-Quinasa/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Receptor de Colecistoquinina B/metabolismo , Células Receptoras Sensoriales/citología , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Ganglios Espinales/citología , Masculino , Ratones , Ratones Endogámicos ICR , Nocicepción/efectos de los fármacos , Dolor/patología , Dolor/fisiopatología , Sincalida/farmacologíaRESUMEN
As an indispensable part of Internet of Things (IoT), wireless sensor networks (WSNs) are more and more widely used with the rapid development of IoT. The neighbor discovery protocols are the premise of communication between nodes and networking in energy-limited self-organizing wireless networks, and play an important role in WSNs. Because the node energy is limited, neighbor discovery must operate in an energy-efficient manner, that is, under the condition of a given energy budget, the neighbor discovery performance should be as good as possible, such that the discovery latency would be as small as possible and the discovered neighbor percentage as large as possible. The indirect neighbor discovery mainly uses the information of the neighbors that have been found by a pairwise discovery method to more efficiently make a re-planning of the discovery wake-up schedules of the original pairwise neighbor discovery, thereby improving the discovery energy efficiency. The current indirect neighbor discovery methods are mainly divided into two categories: one involves removing the inefficient active slots in the original discovery wake-up schedules, and the other involves adding some efficient active slots. However, the two categories of methods have their own limitations. The former does not consider that this removal operation destroys the integrity of the original discovery wake-up schedules and hence the possibility of discovering new neighbors is reduced, which adversely affects the discovered neighbor percentage. For the latter category, there are still inefficient active slots that were not removed in the re-planned wake-up schedules. The motivation of this paper is to combine the advantages of these two types of indirect neighbor discovery methods, that is, to combine the addition of efficient active slots and the removal of inefficient active slots. To achieve this goal, this paper proposes, for the first time, the concept of virtual nodes in neighbor discovery to maximize the integrity of the original wake-up schedules and achieve the goals of adding efficient active slots and removing inefficient active slots. Specifically, a virtual node is a collaborative group that is formed by nodes within a small range. The nodes in a collaborative group share responsibility for the activating task of one member node, and the combination of these nodes' wake-up schedules forms the full wake-up schedule of a node that only uses a pairwise method. In addition, this paper proposes a set of efficient group management mechanisms, and the key steps affecting energy efficiency are analyzed theoretically to obtain the energy-optimal parameters. The extended simulation experiments in multiple scenarios show that, compared with other methods, our neighbor discovery protocol based on virtual nodes (VN-NDP) has a significant improvement in average discovery delay and discovered neighbor percentage performance at a given energy budget. Compared with the typical indirect neighbor discovery algorithm EQS, a neighbor discovery with extended quorum system, our proposed VN-NDP method reduces the average discovery delay by up to 10 . 03 % and increases the discovered neighbor percentage by up to 18 . 35 % .
RESUMEN
BACKGROUND: Migraine is a debilitating neurological disorder involving abnormal trigeminovascular activation and sensitization. However, the underlying cellular and molecular mechanisms remain unclear. METHODS: A rat model of conscious migraine was established through the electrical stimulation (ES) of the dural mater surrounding the superior sagittal sinus. Using patch clamp recording, immunofluorescent labelling, enzyme-linked immunosorbent assays and western blot analysis, we studied the effects of ES on sensory neuronal excitability and elucidated the underlying mechanisms mediated by voltage-gated ion channels. RESULTS: The calcitonin gene-related peptide (CGRP) level in the jugular vein blood and the number of CGRP-positive neurons in the trigeminal ganglia (TGs) were significantly increased in rats with ES-induced migraine. The application of ES increased actional potential firing in both small-sized IB4-negative (IB4-) and IB4+ TG neurons. No significant changes in voltage-gated Na+ currents were observed in the ES-treated groups. ES robustly suppressed the transient outward K+ current (IA) in both types of TG neurons, while the delayed rectifier K+ current remained unchanged. Immunoblot analysis revealed that the protein expression of Kv4.3 was significantly decreased in the ES-treated groups, while Kv1.4 remained unaffected. Interestingly, ES increased the P/Q-type and T-type Ca2+ currents in small-sized IB4- TG neurons, while there were no significant changes in the IB4+ subpopulation of neurons. CONCLUSION: These results suggest that ES decreases the IA in small-sized TG neurons and increases P/Q- and T-type Ca2+ currents in the IB4- subpopulation of TG neurons, which might contribute to neuronal hyperexcitability in a rat model of ES-induced migraine.
Asunto(s)
Estimulación Eléctrica/métodos , Seno Sagital Superior/metabolismo , Ganglio del Trigémino/metabolismo , Potenciales de Acción , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Masculino , Neuronas Aferentes/fisiología , Ratas , Ratas Sprague-Dawley , Seno Sagital Superior/citología , Ganglio del Trigémino/citologíaRESUMEN
Recent studies implicate melatonin in the antinociceptive activity of sensory neurons. However, the underlying mechanisms are still largely unknown. Here, we identify a critical role of melatonin in functionally regulating Cav3.2 T-type Ca2+ channels (T-type channel) in trigeminal ganglion (TG) neurons. Melatonin inhibited T-type channels in small TG neurons via the melatonin receptor 2 (MT2 receptor) and a pertussis toxin-sensitive G-protein pathway. Immunoprecipitation analyses revealed that the intracellular subunit of the MT2 receptor coprecipitated with Gαo . Both shRNA-mediated knockdown of Gαo and intracellular application of QEHA peptide abolished the inhibitory effects of melatonin. Protein kinase C (PKC) antagonists abolished the melatonin-induced T-type channel response, whereas inhibition of conventional PKC isoforms elicited no effect. Furthermore, application of melatonin increased membrane abundance of PKC-eta (PKCη ) while antagonism of PKCη or shRNA targeting PKCη prevented the melatonin-mediated effects. In a heterologous expression system, activation of MT2 receptor strongly inhibited Cav3.2 T-type channel currents but had no effect on Cav3.1 and Cav3.3 current amplitudes. The selective Cav3.2 response was PKCη dependent and was accompanied by a negative shift in the steady-state inactivation curve. Furthermore, melatonin decreased the action potential firing rate of small TG neurons and attenuated the mechanical hypersensitivity in a mouse model of complete Freund's adjuvant-induced inflammatory pain. These actions were inhibited by T-type channel blockade. Together, our results demonstrated that melatonin inhibits Cav3.2 T-type channel activity through the MT2 receptor coupled to novel Gßγ -mediated PKCη signaling, subsequently decreasing the membrane excitability of TG neurons and pain hypersensitivity in mice.
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Canales de Calcio Tipo T/efectos de los fármacos , Melatonina/farmacología , Proteína Quinasa C/metabolismo , Células Receptoras Sensoriales/efectos de los fármacos , Animales , Canales de Calcio Tipo T/metabolismo , Hiperalgesia/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones Endogámicos ICR , Receptor de Melatonina MT2/metabolismo , Células Receptoras Sensoriales/metabolismo , Transducción de Señal/efectos de los fármacos , Ganglio del Trigémino/efectos de los fármacos , Ganglio del Trigémino/metabolismoRESUMEN
Phosphatidylcholine (PC) is a rare membrane lipid in bacteria but crucial for virulence of various plant and animal pathogens. The pcs- mutant lacking PC in bacterial membranes of Pseudomonas syringae pv. syringae van Hall 1336 displayed more ampicillin resistance. Ampicillin susceptibility tests gave an IC50 (half maximal inhibitory concentration) of 52 mg/ml for Pseudomonas syringae pv. syringae van Hall 1336, 53 mg/ml for the complemented strain 1336 RM (pcs-/+) and 90 mg/ml for the 1336 pcs- mutant. Activity assay of ß-lactamase in periplasmic extracts gave 0.050 U/mg for the 1336 wild type, 0.052 U/mg for the 1336RM (pcs-/+), 0.086 U/mg for the 1336 pcs- mutant. Analysis by western blotting showed that the content of AmpC enzyme was markedly different in periplasmic extracts between the wild-type and pcs- mutant strains. Reverse transcriptase PCR also showed that the presence or absence of PC in bacterial membranes did not affect the transcription of ampC gene. The phenotype of the pcs- mutant was able to be recovered to the wild type by introducing a wild-type pcs gene into the pcs- mutant. Similar results were also obtained from the soil-dwelling bacterium Pseudomonas sp. 593. Our results demonstrate that the absence of PC in bacterial membranes facilitates the translocation of Sec-dependent ß-lactamase AmpC from cytoplasm to periplasm, and the enhanced ampicillin-resistance in the pcs- strains mainly comes from effective translocation of AmpC via Sec-pathway.
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Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Citoplasma/metabolismo , Periplasma/metabolismo , Fosfatidilcolinas/metabolismo , Pseudomonas/metabolismo , beta-Lactamasas/metabolismo , Secuencia de Aminoácidos , Resistencia a la Ampicilina , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Secuencia de Bases , Transporte Biológico , Clonación Molecular , Citoplasma/enzimología , ADN Bacteriano , Farmacorresistencia Bacteriana , Pruebas de Enzimas , Regulación Bacteriana de la Expresión Génica , Sueros Inmunes/inmunología , Concentración 50 Inhibidora , Mutación , Periplasma/enzimología , Fosfolípidos , Pseudomonas/efectos de los fármacos , Pseudomonas/enzimología , Pseudomonas/genética , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , beta-Lactamasas/genética , beta-Lactamasas/inmunologíaRESUMEN
OBJECTIVE: This study aimed to reveal the mitochondrial genomes (mtgenomes) of Tetrix japonica and Alulatettix yunnanensis, and the phylogenetics of Orthoptera species. METHODS: The mtgenomes of A. yunnanensis and T. japonica were firstly sequenced and assembled through partial sequences amplification, and then the genome organization and gene arrangement were analyzed. Based on nucleotide/amino acid sequences of 13 protein-coding genes and whole mtgenomes, phylogenetic trees were established on 37 Orthoptera species and 5 outgroups, respectively. RESULTS: Except for a regulation region (A+T rich region), a total of 37 genes were found in mtgenomes of T. japonica and A. yunnanensis, including 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes, which exhibited similar characters with other Orthoptera species. Phylogenetic tree based on 13 concatenated protein-coding nucleotide sequences were considered to be more suitable for phylogenetic reconstruction of Orthoptera species than amino acid sequences and mtgenomes. The phylogenetic relationships of Caelifera species were Acridoidea and Pamphagoidea > Pyrgomorphoidea > Pneumoroidea > Eumastacoidea > Tetrigoidea > Tridactyloidea. Besides, a sister-group relationship between Tettigonioidea and Rhaphidophoroidea was revealed in Ensifera. CONCLUSION: Concatenated protein-coding nucleotide sequences of 13 genes were suitable for reconstruction of phylogenetic relationship in orthopteroid species. Tridactyloidea was a sister group of Tetrigoidea in Caelifera, and Rhaphidophoroidea was a sister group of Tettigonioidea in Ensifera.
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Evolución Molecular , Genoma Mitocondrial/genética , Saltamontes/genética , Animales , Secuencia de Bases , Saltamontes/clasificación , Filogenia , Análisis de Secuencia de ADNRESUMEN
Sinopotamon Bott, 1967 is the most speciose and widely distributed freshwater crab genus in East Asia. Our extensive sampling includes about 76% of the known Sinopotamon taxa, and nearly covers its entire distribution area. Based on mitochondrial cytochrome oxidase I (COI) and 16S rRNA, as well as nuclear 28S rRNA and histone H3, we reconstructed the Sinopotamon phylogeny using maximum likelihood and Bayesian approaches. The divergence time was estimated and multiple methods were used to conduct diversification analyses. The ancestral geographic distribution and character state were reconstructed. Three main clades (Clades I, II and III) that roughly correspond to their main geographic distribution ranges were recovered. Our results challenge the current view of the four major species groups based on the morphological differences in the male first gonopod (G1). The most recent common ancestor of Sinopotamon most likely originated from the Sichuan Basin and surrounding mountains (SBSM) and subsequently dispersed throughout central and eastern China. The exceptionally rapid, recent diversification was detected in Clade II. The high incidence of species-level non-monophyly found in Clade II can be explained by recent rapid radiation. Climatic changes, morphological innovations, range expansion and geographical heterogeneity may all contribute to the diversification in Sinopotamon. This study contributes to our knowledge on diversification of freshwater benthic macro-invertebrates in the East Asian inland ecosystem.
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Braquiuros/clasificación , Agua Dulce , Animales , Núcleo Celular/genética , China , Variación Genética , Geografía , Funciones de Verosimilitud , Mitocondrias/genética , Filogenia , Especificidad de la Especie , Factores de TiempoRESUMEN
AIM: Paenibacillus mucilaginosus (P. mucilaginosus) K02 is implicated in mineral weathering. However, relevant molecular mechanisms remain obscure. The study aims to uncover the bacterium's physiological processes using genomic approaches. METHODS AND RESULTS: Genomic DNA from P. mucilaginosus K02 was sequenced using high-throughput Solexa sequencing technology and then conducted for Clusters of Orthologous Group (COG) annotation. Thereafter, genome sequences of K02 were compared with two strains, 3016 and KNP414. Mummer was applied for collinearity analysis of three P. mucilaginosus genomes. BLAST was used to identify pan and core genes in these strains. Finally, a phylogenetic tree was constructed using the maximum likelihood method by TreeBeST. Complete genome sequence of P. mucilaginosus K02 indicated the strain comprises one circular chromosome with 8,819,200 bases containing 58.3% GC content and 84.75% coding regions. A total of 7299 predicted ORFs were identified in the genome, among them, several genes were related to carbonic anhydrase (CA), and exopolysaccharide biosynthesis and secretion. Moreover, proteins of the predicted genes were annotated in COG categories such as "Carbohydrate transport and metabolism" and "Inorganic ion transport and metabolism." In comparison with KNP414 and 3016, K02 exhibited chromosomal recombination or transposition. A total of 6662 core genes were identified among three P. mucilaginosus strains. The phylogenomic study indicated that P. mucilaginosus K02 was clustered with P. mucilaginosus strains 3016 and KNP414. CONCLUSIONS: In P. mucilaginosus K02, genes related to CA and exopolysaccharide biosynthesis and secretion, and that involved in metabolism-related processes might play significant roles in mineral weathering.
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Genoma Bacteriano , Paenibacillus/genética , Proteínas Bacterianas/genética , Composición de Base , Secuencia de Bases , Cromosomas Bacterianos/genética , Datos de Secuencia Molecular , Paenibacillus/aislamiento & purificación , FilogeniaRESUMEN
Chronic pain represents a prevalent and costly medical challenge globally. Nicotinic acetylcholine receptors (nAChRs), one type of ligand-gated ion channels found extensively in both the central and peripheral nervous systems, have emerged as promising therapeutic targets for chronic pain. Although there are currently no FDA-approved analgesics specifically targeting nAChRs, accumulating preclinical and clinical evidence suggest that selective ligands for alpha 7 (α7) nAChRs show potential for treating chronic pain, boasting a reduced incidence of side effects compared with other nicotinic receptor types. The recent structural resolution of human α7 nAChRs has confirmed their negative association with heightened pain, providing a valuable foundation for the development of targeted medications. This review presents a comprehensive overview, encompassing insights into the roles of α7 nAChRs derived from structural and functional studies, recent advancements in pharmacology, and investigations into their involvement in the pathophysiology of chronic pain. Moreover, the review addresses the variability in analgesic effects based on the type of receptor agonist and highlights the current research limitations. As such, this review offers potential therapeutic approaches for the development of innovative strategies for chronic pain management.
RESUMEN
Heterologous prime-boost has broken the protective immune response bottleneck of the COVID-19 vaccines. however, the underlying mechanisms have not been fully elucidated. Here, we investigated antibody responses and explored the response of germinal center (GC) to priming with inactivated vaccines and boosting with heterologous adenoviral-vectored vaccines or homologous inactivated vaccines in mice. Antibody responses were dramatically enhanced by both boosting regimens. Heterologous immunization induced more robust GC activation, characterized by increased Tfh cell populations and enhanced helper function. Additionally, increased B-cell activation and antibody production were observed in a heterologous regimen. Libra-seq was used to compare the differences of S1-, S2- and NTD-specific B cells between homologous and heterologous vaccination, respectively. S2-specific CD19+ B cells presented increased somatic hypermutations (SHMs), which were mainly enriched in plasma cells. Moreover, a heterologous booster dose promoted the clonal expansion of B cells specific to S2 and NTD regions. In conclusion, the functional role of Tfh and B cells following SARS-CoV-2 heterologous vaccination may be important for modulating antibody responses. These findings provide new insights for the development of SARS-CoV-2 vaccines that induce more robust antibody response.