B-type natriuretic peptide is neither itch-specific nor functions upstream of the GRP-GRPR signaling pathway

BACKGROUND: A recent study by Mishra and Hoon identified B-type natriuretic peptide (BNP) as an important peptide for itch transmission and proposed that BNP activates spinal natriuretic peptide receptor-A (NPRA) expressing neurons, which release gastrin releasing peptide (GRP) to activate GRP receptor (GRPR) expressing neurons to relay itch information from the periphery to the brain (Science 340:968–971, 2013). A central premise for the validity of this novel pathway is the absence of GRP in the dorsal root ganglion (DRG) neurons. To this end, they showed that Grp mRNA in DRG neurons is either absent or barely detectable and claimed that BNP but not GRP is a major neurotransmitter for itch in pruriceptors. They showed that NPRA immunostaining is perfectly co-localized with Grp-eGFP in the spinal cord, and a few acute pain behaviors in Nppb( -/- ) mice were tested. They claimed that BNP is an itch-selective peptide that acts as the first station of a dedicated neuronal pathway comprising a GRP-GRPR cascade for itch. However, our studies, along with the others, do not support their claims. FINDINGS: We were unable to reproduce the immunostaining of BNP and NPRA as shown by Mishra and Hoon. By contrast, we were able to detect Grp mRNA in DRGs using in situ hybridization and real time RT-PCR. We show that the expression pattern of Grp mRNA is comparable to that of GRP protein in DRGs. Pharmacological and genetic blockade of GRP-GRPR signaling does not significantly affect intrathecal BNP-induced scratching behavior. We show that BNP inhibits inflammatory pain and morphine analgesia. CONCLUSIONS: Accumulating evidence demonstrates that GRP is a key neurotransmitter in pruriceptors for mediating histamine-independent itch. BNP-NPRA signaling is involved in both itch and pain and does not function upstream of the GRP-GRPR dedicated neuronal pathway. The site of BNP action in itch and pain and its relationship with GRP remain to be clarified

Cross-inhibition of NMBR and GRPR signaling maintains normal histaminergic itch transmission

We previously showed that gastrin-releasing peptide receptor (GRPR) in the spinal cord is important for mediating nonhistaminergic itch. Neuromedin B receptor (NMBR), the second member of the mammalian bombesin receptor family, is expressed in a largely nonoverlapping pattern with GRPR in the superficial spinal cord, and its role in itch transmission remains unclear. Here, we report that Nmbr knock-out (KO) mice exhibited normal scratching behavior in response to intradermal injection of pruritogens. However, mice lacking both Nmbr and Grpr (DKO mice) showed significant deficits in histaminergic itch. In contrast, the chloroquine (CQ)-evoked scratching behavior of DKO mice is not further reduced compared with Grpr KO mice. These results suggest that NMBR and GRPR could compensate for the loss of each other to maintain normal histamine-evoked itch, whereas GRPR is exclusively required for CQ-evoked scratching behavior. Interestingly, GRPR activity is enhanced in Nmbr KO mice despite the lack of upregulation of Grpr expression; so is NMBR in Grpr KO mice. We found that NMB acts exclusively through NMBR for itch transmission, whereas GRP can signal through both receptors, albeit to NMBR to a much lesser extent. Although NMBR and NMBR(+) neurons are dispensable for histaminergic itch, GRPR(+) neurons are likely to act downstream of NMBR(+) neurons to integrate NMB-NMBR-encoded histaminergic itch information in normal physiological conditions. Together, we define the respective function of NMBR and GRPR in itch transmission, and reveal an unexpected relationship not only between the two receptors but also between the two populations of interneurons in itch signaling

Chronic itch development in sensory neurons requires BRAF signaling pathways

Chronic itch, or pruritus, is associated with a wide range of skin abnormalities. The mechanisms responsible for chronic itch induction and persistence remain unclear. We developed a mouse model in which a constitutively active form of the serine/threonine kinase BRAF was expressed in neurons gated by the sodium channel Nav1.8 (BRAF(Nav1.8) mice). We found that constitutive BRAF pathway activation in BRAF(Nav1.8) mice results in ectopic and enhanced expression of a cohort of itch-sensing genes, including gastrin-releasing peptide (GRP) and MAS-related GPCR member A3 (MRGPRA3), in nociceptors expressing transient receptor potential vanilloid 1 (TRPV1). BRAF(Nav1.8) mice showed de novo neuronal responsiveness to pruritogens, enhanced pruriceptor excitability, and heightened evoked and spontaneous scratching behavior. GRP receptor expression was increased in the spinal cord, indicating augmented coding capacity for itch subsequent to amplified pruriceptive inputs. Enhanced GRP expression and sustained ERK phosphorylation were observed in sensory neurons of mice with allergic contact dermatitis– or dry skin–elicited itch; however, spinal ERK activation was not required for maintaining central sensitization of itch. Inhibition of either BRAF or GRP signaling attenuated itch sensation in chronic itch mouse models. These data uncover RAF/MEK/ERK signaling as a key regulator that confers a subset of nociceptors with pruriceptive properties to initiate and maintain long-lasting itch sensation

Abnormal Global Brain Functional Connectivity in Primary Insomnia Patients: A Resting-State Functional MRI Study

Background: Resting-state functional magnetic resonance imaging (fMRI) studies have uncovered the disruptions of functional brain networks in primary insomnia (PI) patients. However, the etiology and pathogenesis underlying this disorder remains ambiguous, and the insomnia related symptoms are influenced by a complex network organization in the brain. The purpose of this study was to explore the abnormal intrinsic functional hubs in PI patients using a voxel-wise degree centrality (DC) analysis and seed-based functional connectivity (FC) approach.Methods: A total of 26 PI patients and 28 healthy controls were enrolled, and they underwent resting-state fMRI. Degree centrality was measured across the whole brain, and group differences in DC were compared. The peak points, which significantly altered DC between the two groups, were defined as the seed regions and were further used to calculate FC of the whole brain. Later, correlation analyses were performed between the changes in brain function and clinical features.Results: Primary insomnia patients showed DC values lower than healthy controls in the left inferior frontal gyrus (IFG) and middle temporal gyrus (MTG) and showed a higher DC value in the right precuneus. The seed-based analyses demonstrated decreased FC between the left MTG and the left posterior cingulate cortex (PCC), and decreased FC was observed between the right precuneus and the right lateral occipital cortex. Reduced DC in the left IFG and decreased FC in the left PCC were positively correlated with the Pittsburgh sleep quality index and the insomnia severity index.Conclusions: This study revealed that PI patients exhibited abnormal intrinsic functional hubs in the left IFG, MTG, and the right precuneus, as well as abnormal seed-based FC in these hubs. These results contribute to better understanding of how brain function influences the symptoms of PI

Evaluations of oil and gas lateral migration across faults: A case study of Shigezhuang nose structure of Wen'an slope in Baxian sag, Jizhong depression, Bohai Bay Basin, East China

To study the possibility and location of lateral migration of oil and gas across faults, a quantitative evaluation method for lateral migration of oil and gas across faults was established using the Shuangjing Knipe graphic method to identify the juxtaposition site and juxtaposition patterns of sand and sand in the upper and lower walls of the reverse faults and the consequent faults, combined with the oil and gas limit method of fault lateral seal in the test oil area. The quantitative evaluation method was applied to the first and two members of the Paleogene Shahejie strata (referred to as Es1 and Es2) of Shigezhuang nose structure of Wen'an slope in Baxian sag, Jizhong depression, Bohai Bay Basin, to determine the juxtaposition site of sand and sand in the upper and lower walls of the fault, and the shale gouge ratio (SGR) lower values are 26% and 29% respectively in the strata Es1 and Es2. Thus, the location of lateral migration of oil and gas across faults was determined. Based on the oil and gas distribution characteristics of the strata Es1 and Es2, and variation trend of nitrogen compounds in 4 wells in the strata Es1, the results were consistent with the quantitative evaluation of the location of lateral migration of oil and gas across faults, the feasibility of the evaluation method was preliminarily verified. Key words: Bohai Bay Basin, Jizhong depression, Wen'an slope, hydrocarbon lateral migration, shale gouge ratio, fault lateral seal, oil and gas seal limit, Knipe diagram metho

Genetic Diversity of 76 STR Loci in the Dahe Pig

The Dahe pig is one of the representative indigenous pig breeds in the middle-altitude region of southwest China, and has played an important role in the local meat production for a long time. In order to estimate the genetic variability in its population, as well as provide the guidance and basis of genetic background for its conservation and further utilization, 76 microsatellite loci situated on swine’s 19 chromosomes were analyzed for 60 individuals sampled from their natural population by employing PCR and polyacrylamide gel electrophoresis. A total of 347 alleles were detected and all the loci were polymorphic. The number of alleles varied from 3 to 10, with a mean allele and effective allele number of 4.57 and 3.50 at each locus respectively. The mean heterozygosity and polymorphism information content of 76 microsatellite loci were 0.696 2±0.071 6 and 0.644 1±0.091 4, respectively. The results indicated that the genetic diversity of the Dahe pig was rich, and the Dahe pig has a good selection potential for breeding

Spinal apolipoprotein E is involved in inflammatory pain via regulating lipid metabolism and glial activation in the spinal dorsal horn

Abstract Introduction Inflammation and nerve injury promote astrocyte activation, which regulates the development and resolution of pain, in the spinal dorsal horn. APOE regulates lipid metabolism and is predominantly expressed in the astrocytes. However, the effect of astrocytic APOE and lipid metabolism on spinal cellular function is unclear. This study aimed to investigate the effect of spinal Apoe on spinal cellular functions using the complete Freund's adjuvant (CFA)-induced inflammatory pain mouse model. Methods After intraplantar injection of CFA, we assessed pain behaviors in C57BL6 and Apoe knockout (Apoe −/− ) mice using von Frey and Hargreaves’ tests and analyzed dorsal horn samples (L4-5) using western blotting, immunofluorescence, quantitative real-time polymerase chain reaction, and RNA sequencing. Results The Apoe levels were markedly upregulated at 2 h and on days 1 and 3 post-CFA treatment. Apoe was exclusively expressed in the astrocytes. Apoe −/− mice exhibited decreased pain on day 1, but not at 2 h, post-CFA treatment. Apoe −/− mice also showed decreased spinal neuron excitability and paw edema on day 1 post-CFA treatment. Global transcriptomic analysis of the dorsal horn on day 1 post-CFA treatment revealed that the differentially expressed mRNAs in Apoe −/− mice were associated with lipid metabolism and the immune system. Astrocyte activation was impaired in Apoe −/− mice on day 1 post-CFA treatment. The intrathecal injection of Apoe antisense oligonucleotide mitigated CFA-induced pain hypersensitivity. Conclusions Apoe deficiency altered lipid metabolism in astrocytes, exerting regulatory effects on immune response, astrocyte activation, and neuronal activity and consequently disrupting the maintenance of inflammatory pain after peripheral inflammation. Targeting APOE is a potential anti-nociception and anti-inflammatory strategy

Fluorescent DNA-Protected Silver Nanoclusters for Ligand–HIV RNA Interaction Assay

Studying ligand–biomacromolecule interactions provides opportunities for creating new compounds that can efficiently regulate specific biological processes. Ribonucleic acid (RNA) molecules have become attractive drug targets since the discovery of their roles in modulating gene expression, while only a limited number of studies have investigated interactions between ligands and functional RNA molecules, especially those based on nanotechnology. DNA-protected silver nanoclusters (AgNCs) were used to investigate ligand–RNA interactions for the first time in this study. The anthracycline anticancer drug mitoxantrone (MTX) was found to quench the fluorescence of AgNCs. After adding human immunodeficiency virus trans-activation responsive region (TAR) RNA or Rev-response element (RRE) RNA to AgNCs-MTX mixtures, the fluorescence of the AgNCs recovered due to interactions between MTX with RNAs. The binding constants and number of binding sites of MTX to TAR and RRE RNA were determined through theoretical calculations. MTX–RNA interactions were further confirmed in fluorescence polarization and mass spectrometry experiments. The mechanism of MTX-based fluorescence quenching of the AgNCs was also explored. This study provides a new strategy for ligand–RNA binding interaction assay