To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at 27/210 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D 3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at 228 kb that 1,25D-dependently docked VDR, RXR, C/EBPb, and RUNX2. This CRM harbored 3 VDREs and single C/EBPb and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at 27/210 kb and 228 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.-Kaneko, I., Sabir, M. S., Dussik, C. M., Whitfield, G. K., Karrys, A., Hseih, J.-C., Haussler, M. R., Meyer, M. B., Pike, J. W., Jurutka, P. W. 1,25-dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D. FASEB J. 29, 4023-4035 (2015 (1,25D), acts as a classic nuclear receptor ligand that binds specifically to the vitamin D receptor (VDR) to control the transcription of a multitude of genes, primarily those encoding proteins participating in bone mineral metabolism, regulating the immune system, and controlling cell growth and differentiation (1). Liganded VDR attracts one of the retinoid X receptors (RXRs) into a heterodimer that recognizes vitamin D responsive elements (VDREs) in the vicinity of target genes, regulating their expression via the recruitment of comodulator complexes that modify chromatin to effect either induction or repression of the cognate mRNA (2-4). A currently understudied area of vitamin D's function and significance is the CNS, even though the VDR protein is reportedly expressed broadly in the brain, including the neurons and glial cells (5). The most intense immunochemical signal is present in the hypothalamus (paraventricular and supraoptic nuclei and the lateral and ventromedial regions) and in the dopaminergic neurons of the substantia nigra (6). However, VDR protein is also detected in prefrontal cortex, cingulate gyrus, and CA2 region of the hippocampus. Thus, the VDR protein is clearly present in the CNS, but only limited information is available on the functions of VDR and its 1,25D ligand in the bra...

Infection is a devastating complication following an open fracture. We investigated whether local rifampin-loaded hydrogel can combat infection and improve healing in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10-12 weeks and stabilized with an intramedullary pin. A total of 1 × 10 6 colony-forming units (CFU) of MRSA was inoculated. A collagen-based hydrogel containing low-dose (60 μg) and high-dose (300 μg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (μCT), immunohistochemistry, and histological analyses. All untreated MRSA-infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin-1β stain were associated with poor callus formation. High-dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low-dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with highdose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). μCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampinloaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin-loaded hydrogels is required to correlate these findings with clinical efficacy.

Background/AimsIrritable bowel syndrome (IBS) is a multifaceted disorder that afflicts millions of individuals worldwide. IBS is currently diagnosed based on the presence/duration of symptoms and systematic exclusion of other conditions. A more direct manner to identify IBS is needed to reduce healthcare costs and the time required for accurate diagnosis. The overarching objective of this work is to identify gene expression-based biological signatures and biomarkers of IBS. MethodsGene transcripts from 24 tissue biopsy samples were hybridized to microarrays for gene expression profiling. A combination of multiple statistical analyses was utilized to narrow the raw microarray data to the top 200 differentially expressed genes between IBS versus control subjects. In addition, quantitative polymerase chain reaction was employed for validation of the DNA microarray data. Gene ontology/pathway enrichment analysis was performed to investigate gene expression patterns in biochemical pathways. Finally, since vitamin D has been shown to modulate serotonin production in some models, the relationship between serum vitamin D and IBS was investigated via 25-hydroxyvitamin D (25[OH]D) chemiluminescence immunoassay. ResultsA total of 858 genetic features were identified with differential expression levels between IBS and asymptomatic populations. Gene ontology enrichment analysis revealed the serotonergic pathway as most prevalent among the differentially expressed genes. Further analysis via real-time polymerase chain reaction suggested that IBS patient-derived RNA exhibited lower levels of tryptophan hydroxylase-1 expression, the enzyme that catalyzes the rate-limiting step in serotonin biosynthesis. Finally, mean values for 25(OH)D were lower in IBS patients relative to non-IBS controls. Conclusions IntroductionIrritable bowel syndrome (IBS) is one of the most common gastrointestinal (GI) disorders afflicting between 10-15% of the global population.1 Primary symptoms associated with IBS include abdominal pain and irregular bowel movements that, based upon the predominant symptom pattern, may be utilized to subdivide the syndrome into 3 classifications: diarrhea-predominant IBS (IBS-D), constipation-predominant IBS (IBS-C), and mixed IBS (IBS-M).2 However, the subtype of IBS that an individual is classified does not necessarily remain static; patients can evolve over time from diarrheic or mixed type to constipative. The chronic and erratic nature of IBS can lead to a multitude of secondary effects, such as depression, anxiety, poor quality of life, insomnia, and sexual dysfunction. 3Using the current diagnostic model for IBS, the Rome IV criteria, symptoms must be present for 3 months or longer before diagnoses can be established. 4 Other disorders with IBS-like symptoms that should be excluded include celiac disease, inflammatory bowel disease (IBD), malabsorptive disorders, pelvic floor disorders, and colon cancer. Further, once diagnosed, treatment for IBS patients may be mostly palliative in nature with dietary changes...

Bacterial infections involving joints and vital organs represent a challenging clinical problem because of the two concurrent therapeutic goals of bacterial eradication and tissue preservation. In the case of septic arthritis, permanent destruction of articular cartilage by intense host inflammation is commonly seen even after successful treatment of bacterial infection. Here, we provide scientific evidence of a novel treatment modality that can protect articular cartilage and enhanced eradication of causative bacteria in septic arthritis. Locally delivered cell-penetrating antibiotics such as rifampicin effectively eradicate intracellular reservoirs of methicillin-resistant Staphylococcus aureus within joint cells. Furthermore, mitigation of intra-articular inflammation by targeting the NLRP3 (nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3) inflammasome protects articular cartilage from damage in a murine model of knee septic arthritis. Together, concurrent mitigation of intra-articular inflammation and local adjuvant targeting of intracellular bacteria represents a promising new therapeutic strategy for septic arthritis.

The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-D-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1–5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.