P2Y1 Purinergic Receptor Contributes to Remifentanil-Induced Cold Hyperalgesia via Transient Receptor Potential Melastatin 8-Dependent Regulation of N-methyl-d-aspartate Receptor Phosphorylation in Dorsal Root Ganglion.

BACKGROUND: Remifentanil can induce postinfusion cold hyperalgesia. N-methyl-d-aspartate receptor (NMDAR) activation and upregulation of transient receptor potential melastatin 8 (TRPM8) membrane trafficking in dorsal root ganglion (DRG) are critical to cold hyperalgesia derived from neuropathic pain, and TRPM8 activation causes NMDAR-dependent cold response. Contribution of P2Y1 purinergic receptor (P2Y1R) activation in DRG to cold pain hypersensitivity and NMDAR activation induced by P2Y1R upregulation in neurons are also unraveled. This study explores whether P2Y1R contributes to remifentanil-induced cold hyperalgesia via TRPM8-dependent regulation of NMDAR phosphorylation in DRG. METHODS: Rats with remifentanil-induced cold hyperalgesia were injected with TRPM8 antagonist or P2Y1R antagonist at 10 minutes before remifentanil infusion. Cold hyperalgesia (paw lift number and withdrawal duration on cold plate) was measured at -24, 2, 6, 24, and 48 hours following remifentanil infusion. After the last behavioral test, P2Y1R expression, TRPM8 expression and membrane trafficking, and NMDAR subunit (NR1 and NR2B) expression and phosphorylation in DRG were detected by western blot, and colocalization of P2Y1R with TRPM8 was determined by double-labeling immunofluorescence. Two-way repeated measures analysis of variance (ANOVA) or 2 × 2 factorial design ANOVA with repeated measures was used to analyze behavioral data of cold hyperalgesia. One-way ANOVA followed by Bonferroni post hoc comparisons was used to analyze the data in western blot and immunofluorescence. RESULTS: Remifentanil infusion (1 µg·kg-1·min-1 for 60 minutes) induced cold hyperalgesia (hyperalgesia versus control, paw lift number and withdrawal duration on cold plate at 2-48 hours, P < .0001) with upregulated NR1 (hyperalgesia versus naive, 48 hours, mean ± standard deviation [SD], 114.00% ± 12.48% vs 41.75% ± 5.20%, P < .005) and NR2B subunits expression (104.13% ± 8.37% vs 24.63% ± 4.87%, P < .005), NR1 phosphorylation at Ser896 (91.88% ± 7.08% vs 52.00% ± 7.31%, P < .005) and NR2B phosphorylation at Tyr1472 (115.75% ± 8.68% vs 59.75% ± 7.78%, P < .005), TRPM8 expression (115.38% ± 9.27% vs 40.50% ± 4.07%, P < .005) and membrane trafficking (112.88% ± 5.62% vs 48.88% ± 6.49%, P < .005), and P2Y1R expression (128.25% ± 14.86% vs 45.13% ± 7.97%, P < .005) in DRG. Both TRPM8 and P2Y1R antagonists attenuated remifentanil-induced cold hyperalgesia and downregulated increased NR1 and NR2B expression and phosphorylation induced by remifentanil (remifentanil + RQ-00203078 versus remifentanil + saline, NR1 phosphorylation, 69.38% ± 3.66% vs 92.13% ± 4.85%; NR2B phosphorylation, 72.25% ± 6.43% vs 111.75% ± 11.00%, P < .0001). NMDAR activation abolished inhibition of TRPM8 and P2Y1R antagonists on remifentanil-induced cold hyperalgesia. P2Y1R antagonist inhibited remifentanil-evoked elevations in TRPM8 expression and membrane trafficking and P2Y1R-TRPM8 coexpression (remifentanil + 2'-deoxy-N6-methyl adenosine 3',5'-diphosphate [MRS2179] versus remifentanil + saline, coexpression, 8.33% ± 1.33% vs 22.19% ± 2.15%, P < .0001). CONCLUSIONS: Attenuation of remifentanil-induced cold hyperalgesia by P2Y1R inhibition is attributed to downregulations in NMDAR expression and phosphorylation via diminishing TRPM8 expression and membrane trafficking in DRG.

Regulation of nicotinic acetylcholine receptor α3 subtype in adipose tissue dysfunction.

Nicotinic acetylcholine receptor α3 subtype (α3-nAChR) plays a pivotal role in regulating inflammatory responses. Inflammation leads to the adipose tissue dysfunction and further increases the risk of metabolic and cardiovascular diseases. Therefore, we hypothesize that α3-nAChR could regulate the disorder of adipose functions. Adipocytokines and inflammatory cytokines were evaluated in apolipoprotein E knockout (ApoE-/-) mice after α3-nAChR was antagonized and in adipocytes after α3-nAChR gene was silenced. Results showed that in high fat diet-fed ApoE-/- mice with α3-nAChR blocked and in IL-6-stimulated adipocytes with α3-nAChR gene silenced the productions of leptin, resistin, triglyceride, cholesterol and low density lipoprotein were significantly increased but the generations of adiponectin and high density lipoprotein were markedly deceased. Meanwhile, the release of inflammatory cytokines was notably augmented. Moreover, the activation of JAK2/STAT3 was involved in the α3-nAChR-dependent signaling pathways in the regulation of adipose tissue dysfunction. A way may be paved for further investigations for the regulatory role of α3-nAChR in inflammatory and metabolic diseases.

Construction of Multiple Switchable Sensors and Logic Gates Based on Carboxylated Multi-Walled Carbon Nanotubes/Poly(N,N-Diethylacrylamide).

In this work, binary hydrogel films based on carboxylated multi-walled carbon nanotubes/poly(N,N-diethylacrylamide) (c-MWCNTs/PDEA) were successfully polymerized and assembled on a glassy carbon (GC) electrode surface. The electroactive drug probes matrine and sophoridine in solution showed reversible thermal-, salt-, methanol- and pH-responsive switchable cyclic voltammetric (CV) behaviors at the film electrodes. The control experiments showed that the pH-responsive property of the system could be ascribed to the drug components of the solutions, whereas the thermal-, salt- and methanol-sensitive behaviors were attributed to the PDEA constituent of the films. The CV signals particularly, of matrine and sophoridine were significantly amplified by the electrocatalysis of c-MWCNTs in the films at 1.02 V and 0.91 V, respectively. Moreover, the addition of esterase, urease, ethyl butyrate, and urea to the solution also changed the pH of the system, and produced similar CV peaks as with dilution by HCl or NaOH. Based on these experiments, a 6-input/5-output logic gate system and 2-to-1 encoder were successfully constructed. The present system may lead to the development of novel types of molecular computing systems.

A de novo silencer causes elimination of MITF-M expression and profound hearing loss in pigs.

BACKGROUND: Genesis of novel gene regulatory modules is largely responsible for morphological and functional evolution. De novo generation of novel cis-regulatory elements (CREs) is much rarer than genomic events that alter existing CREs such as transposition, promoter switching or co-option. Only one case of de novo generation has been reported to date, in fish and without involvement of phenotype alteration. Yet, this event likely occurs in other animals and helps drive genetic/phenotypic variation. RESULTS: Using a porcine model of spontaneous hearing loss not previously characterized we performed gene mapping and mutation screening to determine the genetic foundation of the phenotype. We identified a mutation in the non-regulatory region of the melanocyte-specific promoter of microphthalmia-associated transcription factor (MITF) gene that generated a novel silencer. The consequent elimination of expression of the MITF-M isoform led to early degeneration of the intermediate cells of the cochlear stria vascularis and profound hearing loss, as well as depigmentation, all of which resemble the typical phenotype of Waardenburg syndrome in humans. The mutation exclusively affected MITF-M and no other isoforms. The essential function of Mitf-m in hearing development was further validated using a knock-out mouse model. CONCLUSIONS: Elimination of the MITF-M isoform alone is sufficient to cause deafness and depigmentation. To our knowledge, this study provides the first evidence of a de novo CRE in mammals that produces a systemic functional effect.

An adjustable Predictive&Prescriptive method for the RO-based optimal power flow problem.

Traditionally before solving the optimal power flow considering uncertainty (OPF-U) problem, the predicted value of uncertainty parameters, such as wind power, e.g., is derived from data using a statistics approach or machine learning. Based on the predicted uncertainty parameters, the solution to the OPF-U problem can be obtained by the prescriptive analytics technique, such as robust optimization (RO). However, it is unclarified how the prediction error in predictive analytics affects solving the OPF-U problem in prescriptive analytics. We propose an adjustable framework method combining machine learning and RO for the OPF-U problem. The k-nearest neighbor is applied to obtain k samples around the predicted value from sufficient historical data. And the optimization results from a minimum volume ellipsoid set containing the k samples are applied to construct KMV set. Then a robust fluctuation region with an adjustable budget level is gained from the KMV set by a two-term exponential formula, which can be embedded into a two-stage RO model. Computational experiments under test cases of different uncertainty scales show the robustness and adjustability of the proposed fluctuation region are better than the state-of-the-art box and ellipsoidal sets. The solution of the proposed two-stage RO model is more economical than the state-of-the-art RO model. The out-of-sample simulation also demonstrates the proposed adjustable Predictive&Prescriptive method can reduce the computational burden as the scale of the system increases when predictive and prescriptive analytics are separated.

Effect and mechanism of apelin on lipopolysaccharide induced acute pulmonary vascular endothelial barrier dysfunction.

Vascular endothelial barrier dysfunction is the most prominent manifestation and important cause of mortality in infectious acute lung injury (ALI). Exogenous apelin is effective in ameliorating lipopolysaccharide (LPS)-induced inflammatory response in ALI lungs, reducing exudation of lung tissue and decreasing mortality. This study set out to investigate the association between apelin and Friend leukemia integration-1 (Fli-1) in the prevention and treatment of ALI, and to elucidate the molecular mechanism by which apelin protects the permeability of the vascular endothelial barrier. At the vivo functional level, lung wet/dry weight ratio was used to detect whole lung permeability, evans blue assay and dual fluorescent protein tracking assay were used to detect lung vascular endothelial permeability, HE staining to observe the inflammatory status of lung tissue, and immunofluorescence staining for VE-cadherin expression levels in blood vessels. The changes in inflammatory factors in bronchoalveolar lavage fluid (BALF) were detected by ELASA. Western blot was used to detect the expression level of proteins. qRT-PCR was performed to detect changes in mRNA expression of Fli-1 and adherent junction-related proteins. The correlation analysis of Fli-1 with vascular endothelial permeability and SRC showed that Fli-1 participated in the process of ALI. After preventive and therapeutic treatment of ALI mice with exogenous apelin, Fli-1, APJ, VE-cadherin, phosphorylated-VE-cadherin (p-VE-cadherin) and ß-catenin were up-regulated, while SRC, phosphorylated-SRC (p-SRC), VEGF and VEGF-R were down-regulated, which indicated that the stability of vascular endothelial barrier was enhanced. With the use of Fli-1 inhibitor irinotecan, the protective effect of apelin was weakened in various functional indexes, genes and proteins. The lung was maintained at the level of the injury. Our research shows that Fli-1 is involved in the LPS-induced ALI process. The molecular mechanism for apelin in preventing endothelial barrier dysfunction in ALI is through up-regulating Fli-1, thus regulating adherens junction-related proteins, and finally recovering the endothelial barrier function.

P2Y1 purinergic receptor inhibition attenuated remifentanil-induced postoperative hyperalgesia via decreasing NMDA receptor phosphorylation in dorsal root ganglion.

BACKGROUND: Remifentanil-induced postoperative hyperalgesia is an intractable side effect of the clinical use of remifentanil, the mechanism of which remains obscure, especially in the peripheral nervous system. N-methyl-D-aspartate receptor (NMDAR) phosphorylation in dorsal root ganglion (DRG) plays a pronociceptive role in neuropathic pain. The contribution of the P2Y1 purinergic receptor (P2Y1R) in DRG to pain hypersensitivity derived from various origins and P2Y1R upregulation-induced NMDAR activation in neurons have also been uncovered. This study aimed to investigate whether P2Y1R participates in nociceptive processing in the DRG and spinal cord in remifentanil-induced postoperative hyperalgesia. METHODS: Rats with remifentanil-induced postoperative hyperalgesia were intrathecally injected with NMDAR antagonist MK801 or P2Y1R antagonist MRS2179 at 10 min prior to remifentanil infusion. Mechanical allodynia, heat hyperalgesia, and cold hyperalgesia were measured at -24 h, 2 h, 6 h, 24 h, and 48 h following remifentanil infusion. The P2Y1R expression and NMDAR expression and phosphorylation in DRG ipsilateral to the incision were detected by Western blot and immunofluorescence. RESULTS: Incision and remifentanil induced mechanical allodynia, heat hyperalgesia, and cold hyperalgesia accompanied by upregulated P2Y1R expression, increased NMDAR subunit NR1 expression and phosphorylation at Ser896, and NR2B expression and phosphorylation at Tyr1472 in DRG. Inhibition of NMDAR phosphorylation by MK801 effectively attenuated remifentanil-induced postoperative hyperalgesia. Furthermore, P2Y1R blockade by MRS2179 not only lessened remifentanil-evoked postoperative hypersensitivity to mechanical, heat, and cold stimuli, but also suppressed the increases in NR1 and NR2B expression and phosphorylation in DRG induced by incision and remifentanil. CONCLUSION: The process by which P2Y1R mediates NMDAR expression and phosphorylation represents a mechanism of remifentanil-induced postoperative hyperalgesia in the DRG and/or spinal cord.