Role of Fractalkine/CX3CR1 Interaction in Light-Induced Photoreceptor Degeneration through Regulating Retinal Microglial Activation and Migration

Background: Excessive exposure to light enhances the progression and severity of some human retinal degenerative diseases. While retinal microglia are likely to be important in neuron damage associated with these diseases, the relationship between photoreceptor damage and microglial activation remains poorly understood. Some recent studies have indicated that the chemokine fractalkine is involved in the pathogenesis of many neurodegenerative diseases. The present study was performed to investigate the cross-talk between injured photoreceptors and activated retinal microglia, focusing on the role of fractalkine and its receptor CX3CR1 in light-induced photoreceptor degeneration. Methodology/Principal Findings: Both in vivo and in vitro experiments were involved in the research. In vivo, Sprague– Dawley rats were exposed to blue light for 24 hours. In vitro, the co-culture of primary retinal microglia and a photoreceptor cell line (661W cell) was exposed to blue light for five hours. Some cultures were pretreated by the addition of anti-CX3CR1 neutralizing antibody or recombinant fractalkine. Expression of fractalkine/CX3CR1 and inflammatory cytokines was detected by immunofluorescence, real-time PCR, Western immunoblot analysis, and ELISA assay. TUNEL method was used to detect cell apoptosis. In addition, chemotaxis assay was performed to evaluate the impact of soluble fractalkine on microglial migration. Our results showed that the expression of fractalkine that was significantly upregulated after exposure to light, located mainly at the photoreceptors. The extent of photoreceptor degeneration and microglial migratio

Transcriptional Regulation of N-Acetylglutamate Synthase

The urea cycle converts toxic ammonia to urea within the liver of mammals. At least 6 enzymes are required for ureagenesis, which correlates with dietary protein intake. The transcription of urea cycle genes is, at least in part, regulated by glucocorticoid and glucagon hormone signaling pathways. N-acetylglutamate synthase (NAGS) produces a unique cofactor, N-acetylglutamate (NAG), that is essential for the catalytic function of the first and rate-limiting enzyme of ureagenesis, carbamyl phosphate synthetase 1 (CPS1). However, despite the important role of NAGS in ammonia removal, little is known about the mechanisms of its regulation. We identified two regions of high conservation upstream of the translation start of the NAGS gene. Reporter assays confirmed that these regions represent promoter and enhancer and that the enhancer is tissue specific. Within the promoter, we identified multiple transcription start sites that differed between liver and small intestine. Several transcription factor binding motifs were conserved within the promoter and enhancer regions while a TATA-box motif was absent. DNA-protein pull-down assays and chromatin immunoprecipitation confirmed binding of Sp1 and CREB, but not C/EBP in the promoter and HNF-1 and NF-Y, but not SMAD3 or AP-2 in the enhancer. The functional importance of these motifs was demonstrated by decreased transcription of reporter constructs following mutagenesis of each motif. The presented data strongly suggest that Sp1, CREB, HNF-1, and NF-Y, that are known to be responsive to hormones and diet, regulate NAGS transcription. This provides molecular mechanism of regulation of ureagenesis in response to hormonal and dietary changes

Differential roles of epigenetic changes and Foxp3 expression in regulatory T cell-specific transcriptional regulation

Naturally occurring regulatory T (Treg) cells, which specifically express the transcription factor forkhead box P3 (Foxp3), are engaged in the maintenance of immunological self-tolerance and homeostasis. By transcriptional start site cluster analysis, we assessed here how genome-wide patterns of DNA methylation or Foxp3 binding sites were associated with Treg-specific gene expression. We found that Treg-specific DNA hypomethylated regions were closely associated with Treg up-regulated transcriptional start site clusters, whereas Foxp3 binding regions had no significant correlation with either up- or down-regulated clusters in nonactivated Treg cells. However, in activated Treg cells, Foxp3 binding regions showed a strong correlation with down-regulated clusters. In accordance with these findings, the above two features of activation-dependent gene regulation in Treg cells tend to occur at different locations in the genome. The results collectively indicate that Treg-specific DNA hypomethylation is instrumental in gene up-regulation in steady state Treg cells, whereas Foxp3 down-regulates the expression of its target genes in activated Treg cells. Thus, the two events seem to play distinct but complementary roles in Treg-specific gene expression

Neuronal Chemokines: Versatile Messengers In Central Nervous System Cell Interaction

Whereas chemokines are well known for their ability to induce cell migration, only recently it became evident that chemokines also control a variety of other cell functions and are versatile messengers in the interaction between a diversity of cell types. In the central nervous system (CNS), chemokines are generally found under both physiological and pathological conditions. Whereas many reports describe chemokine expression in astrocytes and microglia and their role in the migration of leukocytes into the CNS, only few studies describe chemokine expression in neurons. Nevertheless, the expression of neuronal chemokines and the corresponding chemokine receptors in CNS cells under physiological and pathological conditions indicates that neuronal chemokines contribute to CNS cell interaction. In this study, we review recent studies describing neuronal chemokine expression and discuss potential roles of neuronal chemokines in neuron–astrocyte, neuron–microglia, and neuron–neuron interaction

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

Discovery of widespread transcription initiation at microsatellites predictable by sequence-based deep neural network

Using the Cap Analysis of Gene Expression (CAGE) technology, the FANTOM5 consortium provided one of the most comprehensive maps of transcription start sites (TSSs) in several species. Strikingly, ~72% of them could not be assigned to a specific gene and initiate at unconventional regions, outside promoters or enhancers. Here, we probe these unassigned TSSs and show that, in all species studied, a significant fraction of CAGE peaks initiate at microsatellites, also called short tandem repeats (STRs). To confirm this transcription, we develop Cap Trap RNA-seq, a technology which combines cap trapping and long read MinION sequencing. We train sequence-based deep learning models able to predict CAGE signal at STRs with high accuracy. These models unveil the importance of STR surrounding sequences not only to distinguish STR classes, but also to predict the level of transcription initiation. Importantly, genetic variants linked to human diseases are preferentially found at STRs with high transcription initiation level, supporting the biological and clinical relevance of transcription initiation at STRs. Together, our results extend the repertoire of non-coding transcription associated with DNA tandem repeats and complexify STR polymorphism

Duloxetine in patients with diabetic peripheral neuropathic pain in Japan: a randomized, double-blind, noninferiority comparative study with pregabalin

Hiroyuki Enomoto,1 Hitoshi Yasuda,2 Atsushi Nishiyori,3 Shinji Fujikoshi,4 Masashi Furukawa,5 Mitsuhiro Ishida,6 Masashi Takahashi,1 Toshinaga Tsuji,7 Aki Yoshikawa,8 Levent Alev9 1Bio-Medicine, Medicines Development Unit, Eli Lilly Japan K.K, Tokyo, Japan; 2Foundation of Shiga Health Research Center, Shiga, Japan; 3Project Management, Global Development Division, Shionogi & Co. Ltd., Osaka, Japan; 4Statistical Science, Medicines Development Unit, Eli Lilly Japan K.K., Kobe, Japan; 5Biostatistics, Biostatistics Department, Shionogi & Co. Ltd., Osaka, Japan; 6Clinical Research Development, Shionogi & Co. Ltd., Osaka, Japan; 7Medical Affairs Department, Shionogi & Co., Ltd., Osaka, Japan; 8Scientific Communications, Medicines Development Unit, Eli Lilly Japan K.K, Kobe, Japan; 9Eli Lilly, Medical Department, Lilly Turkey, Istanbul, Turkey Purpose: Duloxetine and pregabalin are recommended as first-line treatments for diabetic peripheral neuropathic pain (DPNP). However, studies have not reported a direct comparison between duloxetine and pregabalin. We conducted a postmarketing, randomized, double-blind study to assess the noninferiority of duloxetine compared with pregabalin after 12 weeks of treatment in adult patients with DPNP in Japan (NCT02417935). Patients and methods: Patients (N = 303) with distal symmetrical DPNP were randomized to and were administered duloxetine (40–60 mg/day) or pregabalin (300–600 mg/day). The primary endpoint was the change from baseline in weekly mean of the 24-hour average pain score (numeric rating scale [NRS]). Noninferiority of duloxetine compared with pregabalin was assessed with the primary endpoint at week 12. Secondary measures, including night pain and worst pain, Brief Pain Inventory-Severity and Interference rating short form (BPI-SF), Clinical Global Impression of Improvement (CGI-I), Patient Global Impression of Improvement (PGI-I), and Neuropathic Pain Symptom Inventory (NPSI), health outcome measures (EuroQol 5-Dimension index and VAS), and safety were also assessed. Results: For the 24-hour NRS average pain score, the difference between the duloxetine and pregabalin groups was 0.072 (95% CI: – 0.295, 0.439), and the upper bound of the 95% CI (0.439) did not exceed the predefined noninferiority margin (0.51), at the end of the study period. For secondary outcome measures (night pain, worst pain, BPI-SF, CGI-I, PGI-I, NPSI) and health outcome measures, both the duloxetine and pregabalin treatment groups showed an improvement from baseline with no significant between-group difference. Duloxetine and pregabalin were well tolerated and the safety profiles were consistent with previously reported results. Conclusion: This study demonstrated the noninferior efficacy of duloxetine compared with pregabalin in the treatment of adult patients with DPNP. The safety analyses showed an acceptable tolerability based on safety profiles of duloxetine and pregabalin. Keywords: serotonin and norepinephrine reuptake inhibitor, diabetic peripheral neuropathic pain, duloxetine, Japan, noninferiority, pregabali

Efficacy and safety of controlled-release oxycodone for the management of moderate-to-severe chronic low back pain in Japan: results of an enriched enrollment randomized withdrawal study followed by an open-label extension study

Mikito Kawamata,1 Masako Iseki,2 Mamoru Kawakami,3 Shoji Yabuki,4 Takuma Sasaki,5 Mitsuhiro Ishida,5 Atsushi Nishiyori,5 Hideaki Hida,6 Shin-ichi Kikuchi4 1Department of Anesthesiology and Resuscitology, Shinshu University School of Medicine, Matsumoto, Japan; 2Department of Anesthesiology and Pain Medicine, Juntendo University Faculty of Medicine, Tokyo, Japan; 3Spine Care Center, Wakayama Medical University Kihoku Hospital, Wakayama, Japan; 4Department of Orthopaedic Surgery, Fukushima Medical University School of Medicine, Fukushima, Japan; 5Clinical Development Department, Shionogi & Co. Ltd., Osaka, Japan; 6Biostatistics Center, Shionogi & Co. Ltd., Osaka, Japan Background: Oxycodone is one of the options for the management of CLBP in patients with an inadequate response to other analgesics. However, oxycodone is not yet approved for noncancer pain in Japan. Here, we assessed the efficacy and long-term safety of S-8117, a controlled-release oxycodone formulation, for the management of Japanese CLBP patients. Patients and methods: An initial enriched enrollment randomized withdrawal, double-blind, placebo-controlled, 5-week phase III trial was conducted across 54 centers in Japan to assess the efficacy of S-8117 vs placebo in moderate-to-severe CLBP patients. Subsequently, a 52-week, open-label, single-arm study was conducted across 53 centers in Japan to evaluate the long-term safety of S-8117. The primary endpoint was the time to inadequate analgesic response during 35 days of the double-blind period. Secondary endpoints were the percentages of patients with inadequate analgesic response, discontinuation rate due to inadequate analgesic effects or AEs, and changes in scores of BPI severity, BPI pain interference, SF-36, and Roland-Morris Disability Questionnaire. Safety was assessed as the incidence of AEs and ADRs. Results: Of the 189 patients enrolled in the double-blind study, 130 patients who completed the initial titration period were randomized 1:1 to receive either S-8117 (n=62) or placebo (n=68). Baseline characteristics were comparable across the study groups. The time to inadequate analgesic response was significantly longer in patients treated with S-8117 than placebo (P=0.0095). Secondary endpoints corroborated the efficacy of S-8117 vs placebo. Overall, 478 AEs were reported in 73/75 patients in the long-term study. The most frequent ADRs were somnolence, constipation, and nausea. No case of drug dependence was reported in the long-term study. Conclusion: Short-term efficacy vs placebo and long-term safety of S-8117 were demonstrated for the management of Japanese patients with moderate-to-severe CLBP. Keywords: chronic low back pain, opioids, oxycodone, RC