HDAC6 Inhibition Reverses Cisplatin-Induced Mechanical Hypersensitivity via Tonic Delta Opioid Receptor Signaling.

Peripheral neuropathic pain induced by the chemotherapeutic cisplatin can persist for months to years after treatment. Histone deacetylase 6 (HDAC6) inhibitors have therapeutic potential for cisplatin-induced neuropathic pain since they persistently reverse mechanical hypersensitivity and spontaneous pain in rodent models. Here, we investigated the mechanisms underlying reversal of mechanical hypersensitivity in male and female mice by a 2 week treatment with an HDAC6 inhibitor, administered 3 d after the last dose of cisplatin. Mechanical hypersensitivity in animals of both sexes treated with the HDAC6 inhibitor was temporarily reinstated by a single injection of the neutral opioid receptor antagonist 6ß-naltrexol or the peripherally restricted opioid receptor antagonist naloxone methiodide. These results suggest that tonic peripheral opioid ligand-receptor signaling mediates reversal of cisplatin-induced mechanical hypersensitivity after treatment with an HDAC6 inhibitor. Pointing to a specific role for δ opioid receptors (DORs), Oprd1 expression was decreased in DRG neurons following cisplatin administration, but normalized after treatment with an HDAC6 inhibitor. Mechanical hypersensitivity was temporarily reinstated in both sexes by a single injection of the DOR antagonist naltrindole. Consistently, HDAC6 inhibition failed to reverse cisplatin-induced hypersensitivity when DORs were genetically deleted from advillin+ neurons. Mechanical hypersensitivity was also temporarily reinstated in both sexes by a single injection of a neutralizing antibody against the DOR ligand met-enkephalin. In conclusion, we reveal that treatment with an HDAC6 inhibitor induces tonic enkephalin-DOR signaling in peripheral sensory neurons to suppress mechanical hypersensitivity.SIGNIFICANCE STATEMENT Over one-fourth of cancer survivors suffer from intractable painful chemotherapy-induced peripheral neuropathy (CIPN), which can last for months to years after treatment ends. HDAC6 inhibition is a novel strategy to reverse CIPN without negatively interfering with tumor growth, but the mechanisms responsible for persistent reversal are not well understood. We built on evidence that the endogenous opioid system contributes to the spontaneous, apparent resolution of pain caused by nerve damage or inflammation, referred to as latent sensitization. We show that blocking the δ opioid receptor or its ligand enkephalin unmasks CIPN in mice treated with an HDAC6 inhibitor (latent sensitization). Our work provides insight into the mechanisms by which treatment with an HDAC6 inhibitor apparently reverses CIPN.

HDAC2 in Primary Sensory Neurons Constitutively Restrains Chronic Pain by Repressing α2δ-1 Expression and Associated NMDA Receptor Activity.

α2δ-1 (encoded by the Cacna2d1 gene) is a newly discovered NMDA receptor-interacting protein and is the therapeutic target of gabapentinoids (e.g., gabapentin and pregabalin) frequently used for treating patients with neuropathic pain. Nerve injury causes sustained α2δ-1 upregulation in the dorsal root ganglion (DRG), which promotes NMDA receptor synaptic trafficking and activation in the spinal dorsal horn, a hallmark of chronic neuropathic pain. However, little is known about how nerve injury initiates and maintains the high expression level of α2δ-1 to sustain chronic pain. Here, we show that nerve injury caused histone hyperacetylation and diminished enrichment of histone deacetylase-2 (HDAC2), but not HDAC3, at the Cacna2d1 promoter in the DRG. Strikingly, Hdac2 knockdown or conditional knockout in DRG neurons in male and female mice consistently induced long-lasting mechanical pain hypersensitivity, which was readily reversed by blocking NMDA receptors, inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDA receptor interaction at the spinal cord level. Hdac2 deletion in DRG neurons increased histone acetylation levels at the Cacna2d1 promoter, upregulated α2δ-1 in the DRG, and potentiated α2δ-1-dependent NMDA receptor activity at primary afferent central terminals in the spinal dorsal horn. Correspondingly, Hdac2 knockdown-induced pain hypersensitivity was blunted in Cacna2d1 knockout mice. Thus, our findings reveal that HDAC2 functions as a pivotal transcriptional repressor of neuropathic pain via constitutively suppressing α2δ-1 expression and ensuing presynaptic NMDA receptor activity in the spinal cord. HDAC2 enrichment levels at the Cacna2d1 promoter in DRG neurons constitute a unique epigenetic mechanism that governs acute-to-chronic pain transition.SIGNIFICANCE STATEMENT Excess α2δ-1 proteins produced after nerve injury directly interact with glutamate NMDA receptors to potentiate synaptic NMDA receptor activity in the spinal cord, a prominent mechanism of nerve pain. Because α2δ-1 upregulation after nerve injury is long lasting, gabapentinoids relieve pain symptoms only temporarily. Our study demonstrates for the first time the unexpected role of intrinsic HDAC2 activity at the α2δ-1 gene promoter in limiting α2δ-1 gene transcription, NMDA receptor-dependent synaptic plasticity, and chronic pain development after nerve injury. These findings challenge the prevailing view about the role of general HDAC activity in promoting chronic pain. Restoring the repressive HDAC2 function and/or reducing histone acetylation at the α2δ-1 gene promoter in primary sensory neurons could lead to long-lasting relief of nerve pain.

Association between diet soft drink consumption and metabolic dysfunction-associated steatotic liver disease: findings from the NHANES.

BACKGROUND: Lifestyle change plays a crucial role in the prevention and treatment of metabolic dysfunction-associated steatotic liver disease (MASLD). In recent years, diet soft drinks that emphasize "zero sugar and zero calories" have become all the rage, but whether diet soft drink consumption is associated with MASLD is not clear. METHODS: This study included data from the National Health and Nutrition Examination Surveys (NHANES) in 2003-2006. The assessment of MASLD status primarily relied on the Fatty Liver Index (FLI). Weighted multiple Logistic regression models were constructed to evaluate the association between diet soft drink consumption and MASLD. Additionally, mediation analysis was performed to examine the mediating effect of body mass index (BMI). RESULTS: A total of 2,378 participants were included in the study, among which 1,089 individuals had MASLD, and the weighted prevalence rate was 43.64%. After adjusting for variables related to demographic, lifestyle, and metabolic syndrome, excessive diet soft drink consumption (the "always" frequency) remained significantly associated with the occurrence of MASLD (OR = 1.98, 95%CI = 1.36-2.89, P = 0.003). It was estimated that 84.7% of the total association between diet soft drink consumption and MASLD was mediated by BMI (P < 0.001). CONCLUSIONS: Excessive diet soft drink consumption was associated with the occurrence of MASLD. BMI may play a mediating role in the association between diet soft drink consumption and MASLD.

Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity.

N-methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with GÓ§6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity.SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids.

Identification of crucial lncRNAs and mRNAs in liver regeneration after portal vein ligation through weighted gene correlation network analysis.

BACKGROUND: Portal vein ligation (PVL)-induced liver hypertrophy increases future liver remnant (FLR) volume and improves resectability of large hepatic carcinoma. However, the molecular mechanism by which PVL facilitates liver hypertrophy remains poorly understood. METHODS: To gain mechanistic insight, we established a rat PVL model and carried out a comprehensive transcriptome analyses of hepatic lobes preserving portal blood supply at 0, 1, 7, and 14-day after PVL. The differentially expressed (DE) long-non coding RNAs (lncRNAs) and mRNAs were applied to conduct weighted gene co-expression network analysis (WGCNA). LncRNA-mRNA co-expression network was constructed in the most significant module. The modules and genes associated with PVL-induced liver hypertrophy were assessed through quantitative real-time PCR. RESULTS: A total of 4213 DElncRNAs and 6809 DEmRNAs probesets, identified by transcriptome analyses, were used to carry out WGCNA, by which 10 modules were generated. The largest and most significant module (marked in black_M6) was selected for further analysis. Gene Ontology (GO) analysis of the module exhibited several key biological processes associated with liver regeneration such as complement activation, IL-6 production, Wnt signaling pathway, autophagy, etc. Sixteen mRNAs (Notch1, Grb2, IL-4, Cops4, Stxbp1, Khdrbs2, Hdac2, Gnb3, Gng10, Tlr2, Sod1, Gosr2, Rbbp5, Map3k3, Golga2, and Rev3l) and ten lncRNAs (BC092620, AB190508, EF076772, BC088302, BC158675, BC100646, BC089934, L20987, BC091187, and M23890) were identified as hub genes in accordance with gene significance value, module membership value, protein-protein interaction (PPI) and lncRNA-mRNA co-expression network. Furthermore, the overexpression of 3 mRNAs (Notch1, Grb2 and IL-4) and 4 lncRNAs (BC089934, EF076772, BC092620, and BC088302) was validated in hypertrophic liver lobe tissues from PVL rats and patients undergoing hepatectomy after portal vein embolization (PVE). CONCLUSIONS: Microarray and WGCNA analysis revealed that the 3 mRNAs (Notch1, Grb2 and IL-4) and the 4 lncRNAs (BC089934, EF076772, BC092620 and BC088302) may be promising targets for accelerating liver regeneration before extensive hepatectomy.

Sensory neuron dysfunction in orthotopic mouse models of colon cancer.

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer.

An HDAC6 inhibitor reverses chemotherapy-induced mechanical hypersensitivity via an IL-10 and macrophage dependent pathway.

Chemotherapy-induced peripheral neuropathy (CIPN) impacts a growing number of cancer survivors and treatment options are limited. Histone deacetylase 6 (HDAC6) inhibitors are attractive candidates because they reverse established CIPN and may enhance anti-tumor effects of chemotherapy. Before considering clinical application of HDAC6 inhibitors, the mechanisms underlying reversal of CIPN need to be identified. We showed previously that deletion of Hdac6 from sensory neurons did not prevent cisplatin-induced mechanical hypersensitivity, while global deletion of Hdac6 was protective, indicating involvement of HDAC6 in other cell types. Here we show that local depletion of MRC1 (CD206)-positive macrophages without affecting microglia by intrathecal administration of mannosylated clodronate liposomes reduced the capacity of an HDAC6 inhibitor to reverse cisplatin-induced mechanical hypersensitivity. The HDAC6 inhibitor increased spinal cord Il10 mRNA and this was M2-macrophage dependent. Intrathecal administration of anti-IL-10 antibody or genetic deletion of Il10 prevented resolution of mechanical hypersensitivity. Genetic deletion of the IL-10 receptor from Advillin+ neurons prevented resolution of mechanical hypersensitivity in mice treated with the HDAC6 inhibitor. These findings indicate that treatment with an HDAC6 inhibitor increases macrophage-derived IL-10 signaling to IL-10 receptors on Advillin+ sensory neurons to resolve mechanical hypersensitivity. Cisplatin decreases mitochondrial function in sensory axons, and HDAC6 inhibition can promote axonal transport of healthy mitochondria. Indeed, the HDAC6 inhibitor normalized cisplatin-induced tibial nerve mitochondrial deficits. However, this was independent of macrophages and IL-10 signaling. In conclusion, our findings indicate that administration of an HDAC6 inhibitor reverses cisplatin-induced mechanical hypersensitivity through two complementary pathways: macrophage HDAC6 inhibition to promote IL-10 production and IL-10 signaling to DRG neurons, and neuronal HDAC6 inhibition to restore axonal mitochondrial health.

Age Analysis of Status Updating System with Probabilistic Packet Preemption.

The age of information (AoI) metric was proposed to measure the freshness of messages obtained at the terminal node of a status updating system. In this paper, the AoI of a discrete time status updating system with probabilistic packet preemption is investigated by analyzing the steady state of a three-dimensional discrete stochastic process. We assume that the queue used in the system is Ber/Geo/1/2*/η, which represents that the system size is 2 and the packet in the buffer can be preempted by a fresher packet with probability η. Instead of considering the system's AoI separately, we use a three-dimensional state vector (n,m,l) to simultaneously track the real-time changes of the AoI, the age of a packet in the server, and the age of a packet waiting in the buffer. We give the explicit expression of the system's average AoI and show that the average AoI of the system without packet preemption is obtained by letting η=0. When η is set to 1, the mean of the AoI of the system with a Ber/Geo/1/2* queue is obtained as well. Combining the results we have obtained and comparing them with corresponding average continuous AoIs, we propose a possible relationship between the average discrete AoI with the Ber/Geo/1/c queue and the average continuous AoI with the M/M/1/c queue. For each of two extreme cases where η=0 and η=1, we also determine the stationary distribution of AoI using the probability generation function (PGF) method. The relations between the average AoI and the packet preemption probability η, as well as the AoI's distribution curves in two extreme cases, are illustrated by numerical simulations. Notice that the probabilistic packet preemption may occur, for example, in an energy harvest (EH) node of a wireless sensor network, where the packet in the buffer can be replaced only when the node collects enough energy. In particular, to exhibit the usefulness of our idea and methods and highlight the merits of considering discrete time systems, in this paper, we provide detailed discussions showing how the results about continuous AoI are derived by analyzing the corresponding discrete time system and how the discrete age analysis is generalized to the system with multiple sources. In terms of packet service process, we also propose an idea to analyze the AoI of a system when the service time distribution is arbitrary.

Histone methyltransferase G9a diminishes expression of cannabinoid CB1 receptors in primary sensory neurons in neuropathic pain.

Type 1 cannabinoid receptors (CB1Rs) are expressed in the dorsal root ganglion (DRG) and contribute to the analgesic effect of cannabinoids. However, the epigenetic mechanism regulating the expression of CB1Rs in neuropathic pain is unknown. G9a (encoded by the Ehmt2 gene), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined G9a's role in regulating CB1R expression in the DRG and in CB1R-mediated analgesic effects in an animal model of neuropathic pain. We show that nerve injury profoundly reduced mRNA levels of CB1Rs but increased the expression of CB2 receptors in the rat DRG. ChIP results indicated increased enrichment of histone 3 at lysine 9 dimethylation, a G9a-catalyzed repressive histone mark, at the promoter regions of the CB1R genes. G9a inhibition in nerve-injured rats not only up-regulated the CB1R expression level in the DRG but also potentiated the analgesic effect of a CB1R agonist on nerve injury-induced pain hypersensitivity. Furthermore, in mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce CB1R expression in the DRG and to decrease the analgesic effect of the CB1R agonist. Moreover, nerve injury diminished the inhibitory effect of the CB1R agonist on synaptic glutamate release from primary afferent nerves to spinal cord dorsal horn neurons in WT mice but not in mice lacking Ehmt2 in DRG neurons. Our findings reveal that nerve injury diminishes the analgesic effect of CB1R agonists through G9a-mediated CB1R down-regulation in primary sensory neurons.

Different Crystal Forms of ZnS Nanomaterials for the Adsorption of Elemental Mercury.

ZnS is a promising sorbent in recovering Hg0 from industrial flue gas due to its excellent Hg0 adsorption capacity. However, the internal structure-activity relationship still needs to be further clarified. In this work, ZnS sorbents with different structures were synthesized with the hydrothermal method by tuning the temperature. The samples had significant differences in the crystallinity, morphology, particle size, and sulfur (S) active sites. The results indicated that Hg0 removal performance was determined by the specific surface area and S active sites. ZnS synthesized at low temperatures (80-ZnS and 120-ZnS) had a larger surface area, while the S sites on the high-temperature-synthesized sample (160-ZnS) were more active for Hg0 adsorption. The 160-ZnS sample exhibited a much higher Hg0 adsorption amount per unit surface area. Further characterization revealed that S22- and Sx were the main active sites for Hg0 adsorption. Sx existed in the form of long-chain polysulfur (L-Sx) on 80-ZnS and 120-ZnS, while it exhibited in the form of short-chain polysulfur (S-Sx) on 160-ZnS. L-Sx had negligible adsorption ability, while S-Sx had a high affinity for Hg0. Hg0 can react with S22- and S-Sx, forming α-HgS and ß-HgS, respectively. The new insight in this work can provide theoretical guidance for the design and structure optimization of ZnS, facilitating its practical industrial application.

α-Hederin induces the apoptosis of gastric cancer cells accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

α-Hederin, a monodesmosidic triterpenoid saponin, exhibited promising antitumor potential against a variety of human cancer cell lines. However, few related studies about effects of α-hederin on gastric cancer are available. Herein, our results showed that α-hederin significantly inhibited the proliferation of gastric cancer cells and arrested the cell cycle in G1 phase in vitro (p < .05). Further research of the potential mechanism reflected that α-hederin could induce intracellular glutathione decrement, adenosine triphosphate level, and mitochondrial membrane potential variation via inducing reactive oxygen species accumulation during the apoptosis of gastric cancer cells. Moreover, the detection of mitochondrial and cytosol proteins with apoptosis-inducing factor, apoptosis protease activating factor-1, and cytochrome C showed an increase in the cytosol, followed by a decrease of Bcl-2 levels and increases of caspase-3, caspase-8, caspase-9, and Bax, which revealed that α-hederin induced apoptosis via triggering activation of the mitochondrial pathway. Furthermore, the above changes were amplified when pretreated with buthionine sulfoximine, whereas attenuated in the group pretreated with NAC than α-hederin alone (p < .05). In addition, α-hederin significantly inhibited the growth of xenografted gastric tumors with favorable safety. In conclusion, α-hederin could inhibit the proliferation and induce apoptosis of gastric cancer accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway.

RE1-silencing transcription factor controls the acute-to-chronic neuropathic pain transition and Chrm2 receptor gene expression in primary sensory neurons.

Neuropathic pain is associated with persistent changes in gene expression in primary sensory neurons, but the underlying epigenetic mechanisms that cause these changes remain unclear. The muscarinic cholinergic receptors (mAChRs), particularly the M2 subtype (encoded by the cholinergic receptor muscarinic 2 (Chrm2) gene), are critically involved in the regulation of spinal nociceptive transmission. However, little is known about how Chrm2 expression is transcriptionally regulated. Here we show that nerve injury persistently increased the expression of RE1-silencing transcription factor (REST, also known as neuron-restrictive silencing factor [NRSF]), a gene-silencing transcription factor, in the dorsal root ganglion (DRG). Remarkably, nerve injury-induced chronic but not acute pain hypersensitivity was attenuated in mice with Rest knockout in DRG neurons. Also, siRNA-mediated Rest knockdown reversed nerve injury-induced chronic pain hypersensitivity in rats. Nerve injury persistently reduced Chrm2 expression in the DRG and diminished the analgesic effect of muscarine. The RE1 binding site on the Chrm2 promoter is required for REST-mediated Chrm2 repression, and nerve injury increased the enrichment of REST in the Chrm2 promoter in the DRG. Furthermore, Rest knockdown or genetic ablation in DRG neurons normalized Chrm2 expression and augmented muscarine's analgesic effect on neuropathic pain and fully reversed the nerve injury-induced reduction in the inhibitory effect of muscarine on glutamatergic input to spinal dorsal horn neurons. Our findings indicate that nerve injury-induced REST up-regulation in DRG neurons plays an important role in the acute-to-chronic pain transition and is essential for the transcriptional repression of Chrm2 in neuropathic pain.

Increased α2δ-1-NMDA receptor coupling potentiates glutamatergic input to spinal dorsal horn neurons in chemotherapy-induced neuropathic pain.

Painful peripheral neuropathy is a severe and difficult-to-treat neurological complication associated with cancer chemotherapy. Although chemotherapeutic drugs such as paclitaxel are known to cause tonic activation of presynaptic NMDA receptors (NMDARs) to potentiate nociceptive input, the molecular mechanism involved in this effect is unclear. α2δ-1, commonly known as a voltage-activated calcium channel subunit, is a newly discovered NMDAR-interacting protein and plays a critical role in NMDAR-mediated synaptic plasticity. Here we show that paclitaxel treatment in rats increases the α2δ-1 expression level in the dorsal root ganglion and spinal cord and the mRNA levels of GluN1, GluN2A, and GluN2B in the spinal cord. Paclitaxel treatment also potentiates the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. Strikingly, inhibiting α2δ-1 trafficking with pregabalin, disrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus-interfering peptide, or α2δ-1 genetic ablation fully reverses paclitaxel treatment-induced presynaptic NMDAR-mediated glutamate release from primary afferent terminals to spinal dorsal horn neurons. In addition, intrathecal injection of pregabalin or α2δ-1 C-terminus-interfering peptide and α2δ-1 knockout in mice markedly attenuate paclitaxel-induced pain hypersensitivity. Our findings indicate that α2δ-1 is required for paclitaxel-induced tonic activation of presynaptic NMDARs at the spinal cord level. Targeting α2δ-1-bound NMDARs, not the physiological α2δ-1-free NMDARs, may be a new strategy for treating chemotherapy-induced neuropathic pain. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Streptozotocin-Induced Diabetic Neuropathic Pain Is Associated with Potentiated Calcium-Permeable AMPA Receptor Activity in the Spinal Cord.

Neuronal hyperactivity in the spinal dorsal horn can amplify nociceptive input in diabetic neuropathic pain. The glutamate N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDA receptors and AMPA receptors, respectively) are involved in spinal nociceptive transmission. It is unclear, however, whether painful diabetic neuropathy is associated with changes in the activity of synaptic NMDA receptors and AMPA receptors in spinal dorsal horn neurons. AMPA receptors lacking GluA2 are Ca2+-permeable (CP-AMPA receptors), and their currents display characteristic inward rectification. In this study, we showed that evoked excitatory postsynaptic currents (EPSCs), induced by streptozotocin, exhibited inward rectification in spinal dorsal neurons in diabetic rats. Presynaptic and postsynaptic NMDA receptor activity in the spinal dorsal horn was similar in diabetic and control rats. In the dorsal spinal cord, the membrane GluA2 protein level was significantly lower in diabetic than in control rats, whereas the cytosolic GluA2 level was greater in diabetic than in control rats. In contrast, the GluA1 subunit levels in the plasma membrane and cytosol did not differ between the two groups. Blocking CP-AMPA receptors significantly reduced the amplitude of EPSCs of dorsal horn neurons in diabetic but not in control rats. Furthermore, blocking spinal CP-AMPA receptors reduced pain hypersensitivity in diabetic rats but had no effect on nociception in control rats. Our study suggests that diabetic neuropathy augments CP-AMPA receptor activity in the spinal dorsal horn by causing intracellular retention of GluA2 and impairing GluA2 membrane trafficking. Increased prevalence of spinal CP-AMPA receptors sustains diabetic neuropathic pain. SIGNIFICANCE STATEMENT: This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition.

α-Hederin Induces Apoptosis of Esophageal Squamous Cell Carcinoma via an Oxidative and Mitochondrial-Dependent Pathway.

BACKGROUND: α-Hederin has been shown promising anti-tumor potential against various cancer cell lines. However, reports about effects of α-hederin on esophageal squamous cell carcinoma (ESCC) are still unavailable. AIM: To investigate the inhibitory effects of α-hederin on ESCC and explore the underlying mechanism. METHODS: Human esophageal carcinoma cell line (Eca-109) was used for the experiment. Cell Counting Kit-8, flow cytometry, Hoechst 33258 staining, enhanced ATP assay kit, 2',7'-dichlorofluorescin diacetate, JC-1 kit, and Western bolt were used to assess the cell viability, cycle, apoptosis, cellular ATP content, reactive oxygen species (ROS) level, mitochondrial membrane potential (MMP), and protein expression, respectively, in vitro. Xenografted tumor model was constructed to evaluate the in vivo anti-tumor effects of α-hederin. RESULTS: Compared with control group, α-hederin significantly inhibited the proliferation, induced apoptosis of ESCC, and arrested the cell cycle in G1 phase (P < 0.05). α-Hederin induced the accumulation of ROS, decrement of ATP levels, and disruption of MMP (P < 0.05). The detection of mitochondrial and cytosol proteins showed that AIF, Apaf-1, and Cyt C were released and increased in cytoplasm, and then, caspase-3, caspase-9, and Bax were involved and increased, while Bcl-2 level was decreased (P < 0.05). Furthermore, the above changes were amplified in the group pretreated with L-buthionine sulfoximine, while N-acetyl-L-cysteine plays an opposite role (P < 0.05). Meanwhile, α-hederin significantly inhibited the growth of xenografted tumors with favorable safety. CONCLUSION: α-Hederin could inhibit the proliferation and induce apoptosis of ESCC via dissipation of the MMP with simultaneous ROS generation and activation of the mitochondrial pathway.

In-situ graft-crosslinked gold nanoparticles with high-density surface defects and coated with a polytaurine membrane for the voltammetric determination of dopamine.

Well-dispersed and graft-crosslinked gold nanoparticles (AuNPs) were synthesized by the reduction of tetrachloroaurate with hydrazine at room temperature. The AuNPs possess a high density of surface defects which is due to grafting of n-octanoic acid to polyvinylpyrrolidone. The physical and chemical properties of the resulting AuNPs were characterized by UV-vis, XRD, TEM/HRTEM, SAED, and XPS, respectively. The modified AuNPs were placed on a glassy carbon electrode (GCE) in an electropolymerized taurine layer to obtain a sensitive, selective, stable and rapid electrochemical dopamine sensor. The peak current, typically measured at 0.17 V (vs. SCE), increases linearly in the 1.0 to 120 µM dopamine concentration range, and the limit of detection (at S/N = 3) is 0.16 µM with a sensitivity of 2.94 µA·µM-1·cm-2. The sensor was successfully applied to the determination of dopamine in injections and spiked serum samples. The recoveries from spiked serum samples range from 97.5 to 102.4%, with RSDs ranging between 2.8 and 3.4%. Graphical abstract Schematic representation of a glassy carbon electrode modified with in-situ graft-crosslinked gold nanoparticles combined with an electropolymerized polytaurine membrane. The sensor exhibits excellent features towards dopamine determination.

CaMKII Regulates Synaptic NMDA Receptor Activity of Hypothalamic Presympathetic Neurons and Sympathetic Outflow in Hypertension.

NMDAR activity in the hypothalamic paraventricular nucleus (PVN) is increased and critically involved in heightened sympathetic vasomotor tone in hypertension. Calcium/calmodulin-dependent protein kinase II (CaMKII) binds to and modulates NMDAR activity. In this study, we determined the role of CaMKII in regulating NMDAR activity of PVN presympathetic neurons in male spontaneously hypertensive rats (SHRs). NMDAR-mediated EPSCs and puff NMDA-elicited currents were recorded in spinally projecting PVN neurons in SHRs and male Wistar-Kyoto (WKY) rats. The basal amplitude of evoked NMDAR-EPSCs and puff NMDA currents in retrogradely labeled PVN neurons were significantly higher in SHRs than in WKY rats. The CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP) normalized the increased amplitude of NMDAR-EPSCs and puff NMDA currents in labeled PVN neurons in SHRs but had no effect in WKY rats. Treatment with AIP also normalized the higher frequency of NMDAR-mediated miniature EPSCs of PVN neurons in SHRs. CaMKII-mediated phosphorylation level of GluN2B serine 1303 (S1303) in the PVN, but not in the hippocampus and frontal cortex, was significantly higher in SHRs than in WKY rats. Lowering blood pressure with celiac ganglionectomy in SHRs did not alter the increased level of phosphorylated GluN2B S1303 in the PVN. In addition, microinjection of AIP into the PVN significantly reduced arterial blood pressure and lumbar sympathetic nerve discharges in SHRs. Our findings suggest that CaMKII activity is increased in the PVN and contributes to potentiated presynaptic and postsynaptic NMDAR activity to elevate sympathetic vasomotor tone in hypertension.SIGNIFICANCE STATEMENT Heightened sympathetic vasomotor tone is a major contributor to the development of hypertension. Although glutamate NMDA receptor (NMDAR)-mediated excitatory drive in the hypothalamus plays a critical role in increased sympathetic output in hypertension, the molecular mechanism involved in potentiated NMDAR activity of hypothalamic presympathetic neurons remains unclear. Here we show that the activity of calcium/calmodulin-dependent protein kinase II (CaMKII) is increased and plays a key role in the potentiated presynaptic and postsynaptic NMDAR activity of hypothalamic presympathetic neurons in hypertension. Also, the inhibition of CaMKII in the hypothalamus reduces elevated blood pressure and sympathetic nerve discharges in hypertension. This new knowledge extends our understanding of the mechanism of synaptic plasticity in the hypothalamus and suggests new strategies to treat neurogenic hypertension.

Focal Cerebral Ischemia and Reperfusion Induce Brain Injury Through α2δ-1-Bound NMDA Receptors.

Background and Purpose- Glutamate NMDARs (N-methyl-D-aspartate receptors) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. α2δ-1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of α2δ-1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods- Mice were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results- Middle cerebral artery occlusion increased α2δ-1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the α2δ-1-NMDAR physical interaction in the mouse brain tissue. Inhibiting α2δ-1 with gabapentin, uncoupling the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or α2δ-1 C-terminus-interfering peptide or Cacna2d1 genetic knock-out reduced middle cerebral artery occlusion-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions- α2δ-1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and α2δ-1-bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting α2δ-1-bound NMDARs, without impairing physiological α2δ-1-free NMDARs, may be a promising strategy for treating ischemic stroke.

Thymoquinone Augments Cisplatin-Induced Apoptosis on Esophageal Carcinoma Through Mitigating the Activation of JAK2/STAT3 Pathway.

BACKGROUND: Thymoquinone (TQ) is the major constituent of Nigella sativa seed and has shown biological activity in various human carcinomas. However, few studies have reported its effect on esophageal carcinoma (EC). AIMS: To explore the chemosensitive effect and mechanism of TQ in augmentation of cisplatin (DDP)-induced apoptosis of EC, both in vitro and in vivo. METHODS: The viability and apoptosis of esophageal carcinoma cells were detected by the Cell Counting Kit-8 assay, flow cytometry, and Hoechst 33258 staining. The expression levels of JAK2, p-JAK2, STAT3, p-STAT3, Bax, Bcl-2, Cyclin D1, Survivin, and caspase-3, 7, 9 were evaluated by western blot analysis. The histological changes were examined by TUNEL technique and immunohistochemical analysis. RESULTS: TQ enhanced the proapoptotic effect of DDP in human esophageal carcinoma cell line Eca-109, while blocking the activation of JAK2/STAT3 signaling pathway. The apoptosis of esophageal carcinoma cells was induced via blocking the activation of JAK2/STAT3 by using a molecular inhibitor (WP1066). Consistent with the in vivo and in vitro results, TQ increased cellular apoptosis and enriched the chemosensitivity of DDP. CONCLUSIONS: TQ along with DDP may regulate the progression of EC and has potential to be a chemotherapeutic agent in EC.

Requirement for flap endonuclease 1 (FEN1) to maintain genomic stability and transcriptional gene silencing in Arabidopsis.

As a central component in the maturation of Okazaki fragments, flap endonuclease 1 (FEN1) removes the 5'-flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. The Arabidopsis fen1-1 mutant is hypersensitive to methyl methanesulfonate and shows reduced telomere length. Interestingly, genome-wide chromatin immunoprecipitation and RNA sequencing results demonstrate that FEN1 mutation leads to a decrease in the level of H3K27me3 and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS as well as maintaining genome stability in Arabidopsis.