GnRH peripherally modulates nociceptor functions, exacerbating mechanical pain.

The function of peripheral nociceptors, the neurons that relay pain signals to the brain, are frequently tuned by local and systemic modulator substances. In this context, neurohormonal effects are emerging as an important modulatory mechanism, but many aspects remain to be elucidated. Here we report that gonadotropin-releasing hormone (GnRH), a brain-specific neurohormone, can aggravate pain by acting on nociceptors in mice. GnRH and GnRHR, the receptor for GnRH, are expressed in a nociceptor subpopulation. Administration of GnRH and its analogue, localized for selectively affecting the peripheral neurons, deteriorated mechanical pain, which was reproducible in neuropathic conditions. Nociceptor function was promoted by GnRH treatment in vitro, which appears to involve specific sensory transient receptor potential ion channels. These data suggest that peripheral GnRH can positively modulate nociceptor activities in its receptor-specific manner, contributing to pain exacerbation. Our study indicates that GnRH plays an important role in neurohormonal pain modulation via a peripheral mechanism.

Gpr83 Tunes Nociceptor Function, Controlling Pain.

The function of peripheral nociceptors is frequently tuned by the action of G protein-coupled receptors (GPRs) that are expressed in them, which contribute to pain alteration. Expanding new information on such GPRs and predicting their potential outcomes can help to construct new analgesic strategies based on their modulations. In this context, we attempted to present a new GPR not yet acknowledged for its pain association. Gpr83 exhibits relatively high expressions in the peripheral nervous system compared to other tissues when we mined and reconstructed Gene Expression Omnibus (GEO) metadata, which we confirmed using immunohistochemistry on murine dorsal root ganglia (DRG). When Gpr83 expression was silenced in DRG, neuronal and behavioral nociception were all downregulated. Pathologic pain in hind paw inflammation and chemotherapy-induced peripheral neuropathy were also alleviated by this Gpr83 knockdown. Dependent on exposure time, the application of a known endogenous Gpr83 ligand PEN showed differential effects on nociceptor responses in vitro. Localized PEN administration mitigated pain in vivo, probably following Gq/11-involved GPR downregulation caused by the relatively constant exposure. Collectively, this study suggests that Gpr83 action contributes to the tuning of peripheral pain sensitivity and thus indicates that Gpr83 can be among the potential GPR targets for pain modulation.

Multidetector CT Findings of Acquired Spondylolysis and Spondylolisthesis after Posterior Lumbar Laminectomy.

Purpose: We aimed to analyze postoperative multidetector CT (MDCT) of acquired spondylolysis and spondylolisthesis after posterior lumbar laminectomy. Materials and Methods: We enrolled 74 patients, from 2003 to 2017, who underwent posterior lumbar laminectomy with both pre and postoperative MDCT. The patients were categorized into the following two groups: group 1 without fusion and group 2 with fusion. We analyzed laminectomy width, level and location of spondylolysis or spondylolisthesis, facet changes, and fatty infiltration of paraspinal muscles on postoperative MDCT. Results: Incidence of spondylolysis or spondylolisthesis was 4 of 20 patients in group 1 and 2 of 54 patients in group 2. The laminectomy width (%) was defined as the percentage of the width of laminectomy to total lamina length. Mean laminectomy width (%) in patients with spondylolysis or spondylolisthesis was 54.0 in group 1 and 53.2 in group 2, in contrast to that in patients without spondylolysis or spondylolisthesis, which was 35.0 in group 1. The spondylolysis was observed at the level of the laminectomy and below pars interarticularis in group 1 and below the fusion mass at isthmic region in group 2. Conclusion: MDCT facilitates the diagnosis of postsurgical acquired spondylolysis and spondylolisthesis and demonstrates typical location of spondylolysis. Greater laminectomy width has been associated with occurrence of acquired spondylolysis and spondylolisthesis.

Optimized Methods for the Isolation of Arabidopsis Female Central Cells and Their Nuclei.

The Arabidopsis female gametophyte contains seven cells with eight haploid nuclei buried within layers of sporophytic tissue. Following double fertilization, the egg and central cells of the gametophyte develop into the embryo and endosperm of the seed, respectively. The epigenetic status of the central cell has long presented an enigma due both to its inaccessibility, and the fascinating epigenome of the endosperm, thought to have been inherited from the central cell following activity of the DEMETER demethylase enzyme, prior to fertilization. Here, we present for the first time, a method to isolate pure populations of Arabidopsis central cell nuclei. Utilizing a protocol designed to isolate leaf mesophyll protoplasts, we systematically optimized each step in order to efficiently separate central cells from the female gametophyte. We use initial manual pistil dissection followed by the derivation of central cell protoplasts, during which process the central cell emerges from the micropylar pole of the embryo sac. Then, we use a modified version of the Isolation of Nuclei TAgged in specific Cell Types (INTACT) protocol to purify central cell nuclei, resulting in a purity of 75-90% and a yield sufficient to undertake downstream molecular analyses. We find that the process is highly dependent on the health of the original plant tissue used, and the efficiency of protoplasting solution infiltration into the gametophyte. By isolating pure central cell populations, we have enabled elucidation of the physiology of this rare cell type, which in the future will provide novel insights into Arabidopsis reproduction.

Control of Paternally Expressed Imprinted UPWARD CURLY LEAF1, a Gene Encoding an F-Box Protein That Regulates CURLY LEAF Polycomb Protein, in the Arabidopsis Endosperm.

Genomic imprinting, an epigenetic process in mammals and flowering plants, refers to the differential expression of alleles of the same genes in a parent-of-origin-specific manner. In Arabidopsis, imprinting occurs primarily in the endosperm, which nourishes the developing embryo. Recent high-throughput sequencing analyses revealed that more than 200 loci are imprinted in Arabidopsis; however, only a few of these imprinted genes and their imprinting mechanisms have been examined in detail. Whereas most imprinted loci characterized to date are maternally expressed imprinted genes (MEGs), PHERES1 (PHE1) and ADMETOS (ADM) are paternally expressed imprinted genes (PEGs). Here, we report that UPWARD CURLY LEAF1 (UCL1), a gene encoding an E3 ligase that degrades the CURLY LEAF (CLF) polycomb protein, is a PEG. After fertilization, paternally inherited UCL1 is expressed in the endosperm, but not in the embryo. The expression pattern of a ß-glucuronidase (GUS) reporter gene driven by the UCL1 promoter suggests that the imprinting control region (ICR) of UCL1 is adjacent to a transposable element in the UCL1 5'-upstream region. Polycomb Repressive Complex 2 (PRC2) silences the maternal UCL1 allele in the central cell prior to fertilization and in the endosperm after fertilization. The UCL1 imprinting pattern was not affected in paternal PRC2 mutants. We found unexpectedly that the maternal UCL1 allele is reactivated in the endosperm of Arabidopsis lines with mutations in cytosine DNA METHYLTRANSFERASE 1 (MET1) or the DNA glycosylase DEMETER (DME), which antagonistically regulate CpG methylation of DNA. By contrast, maternal UCL1 silencing was not altered in mutants with defects in non-CpG methylation. Thus, silencing of the maternal UCL1 allele is regulated by both MET1 and DME as well as by PRC2, suggesting that divergent mechanisms for the regulation of PEGs evolved in Arabidopsis.

The catalytic subunit of Arabidopsis DNA polymerase α ensures stable maintenance of histone modification.

Mitotic inheritance of identical cellular memory is crucial for development in multicellular organisms. The cell type-specific epigenetic state should be correctly duplicated upon DNA replication to maintain cellular memory during tissue and organ development. Although a role of DNA replication machinery in maintenance of epigenetic memory has been proposed, technical limitations have prevented characterization of the process in detail. Here, we show that INCURVATA2 (ICU2), the catalytic subunit of DNA polymerase α in Arabidopsis, ensures the stable maintenance of repressive histone modifications. The missense mutant allele icu2-1 caused a defect in the mitotic maintenance of vernalization memory. Although neither the recruitment of CURLY LEAF (CLF), a SET-domain component of Polycomb Repressive Complex 2 (PRC2), nor the resultant deposition of the histone mark H3K27me3 required for vernalization-induced FLOWERING LOCUS C (FLC) repression were affected, icu2-1 mutants exhibited unstable maintenance of the H3K27me3 level at the FLC region, which resulted in mosaic FLC de-repression after vernalization. ICU2 maintains the repressive chromatin state at additional PRC2 targets as well as at heterochromatic retroelements. In icu2-1 mutants, the subsequent binding of LIKE-HETEROCHROMATIN PROTEIN 1 (LHP1), a functional homolog of PRC1, at PRC2 targets was also reduced. We demonstrated that ICU2 facilitates histone assembly in dividing cells, suggesting a possible mechanism for ICU2-mediated epigenetic maintenance.

Identification of regulators required for the reactivation of FLOWERING LOCUS C during Arabidopsis reproduction.

FLOWERING LOCUS C (FLC) is a central floral repressor for the determination of flowering time in Arabidopsis. FLC expression is reactivated upon fertilization and regulated during seed development to ensure the appropriate floral behavior; however, the molecular mechanism for this process is largely unknown. Here, we report the identification of crucial regulators for FLC reactivation during embryogenesis by analyzing FLC::GUS and endogenous FLC expression. We newly define that the full reactivation of FLC requires a FRIGIDA (FRI)-containing protein complex throughout embryogenesis. Mutations in EARLY FLOWERING 7 (ELF7) and VERNALIZATION INDEPENDENCE4 (VIP4) showed severe defects in the reactivation of FLC transcription, suggesting that both of the genes, Arabidopsis homologs of the members of the yeast RNA polymerase II-associated factor 1 (Paf1) complex, are indispensable for FLC reactivation. actin-related protein 6 (arp6), arabidopsis trithorax 1 (atx1), arabidopsis trithorax-related 7 (atxr7), and atx1 atxr7 double mutants also caused the downregulation of FLC during seed development, but the defects were less severe than those in mutants for the FRI- and Paf1-complexes. These results suggest that the ARP6-containing Swr1-complex and FLC-specific histone methyltransferases, ATX1 and ATXR7, have relatively partial roles in FLC reactivation. In contrast to the roles of the histone modifiers, factors in the DNA methylation pathway and biogenesis of small RNAs are not involved in FLC regulation during reproduction. Taken together, our results demonstrate that adjustment by select FLC activators is critical for the re-establishment of an FLC expression state after fertilization to ensure competence for optimal flowering in the next generation.