Combination of naproxen and a chemically-stable eicosapentaenoic acid analog provide additive tumor protection in Pirc rats.

Colorectal cancer (CRC) is the second leading cause of cancer-related deaths in the United States. Patients with the genetic disorder Familial Adenomatous Polyposis (FAP) develop hundreds to thousands of polyps that unless removed by prophylactic colectomy will progress to CRC at an early age. Nonsteroidal anti-inflammatory drugs (NSAIDs) and the ω-3 polyunsaturated fatty acid (PUFA) eicosapentaenoic acid (EPA), have been evaluated for their chemopreventive potential in delaying CRC onset in high-risk patients. In our study, we determined whether the NSAID, naproxen, alone or in combination with a chemically-stable EPA analog (TP-252), affects tumor formation in the ApcPirc rat model. When compared to control diet, animals fed naproxen or HD TP-252 had 66% and 82% fewer tumors, respectively. However, animals fed a combination of naproxen and HD TP-252, exhibited a 95% reduction in tumor formation and a 98% reduction in tumor volume, respectively. To elucidate potential mechanisms of tumor protection, a comprehensive, targeted lipidomic analysis was performed on colonic mucosa to determine changes in eicosanoid metabolism. Animals receiving TP-252 alone or in combination with naproxen had significantly reduced mucosal levels of proinflammatory ω-6 eicosanoids (PGE2 , 5-HETE and 14,15-DiHETrE), along with a simultaneous increase in anti-inflammatory EPA-derived ω-3 eicosanoids. A comprehensive lipidomic analysis also uncovered several potential pharmacodynamic (PD) lipid biomarkers, including resolvin E2, 9-HEPE, 12-HEPE and 18-HEPE, that were significantly correlated with tumor protection. Further studies with this drug combination should be focused on dose optimization and the role of EPA-derived lipid mediators in CRC initiation and progression.

Cultivation of common bacterial species and strains from human skin, oral, and gut microbiota.

BACKGROUND: Genomics-driven discoveries of microbial species have provided extraordinary insights into the biodiversity of human microbiota. In addition, a significant portion of genetic variation between microbiota exists at the subspecies, or strain, level. High-resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. High-throughput approaches are needed to acquire and identify the significant species- and strain-level diversity present in the oral, skin, and gut microbiome. Here, we describe and validate a streamlined workflow for cultivating dominant bacterial species and strains from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling. RESULTS: Of total genera discovered by either metagenomic sequencing or culturomics, our cultivation pipeline recovered between 18.1-44.4% of total genera identified. These represented a high proportion of the community composition reconstructed with metagenomic sequencing, ranging from 66.2-95.8% of the relative abundance of the overall community. Fourier-Transform Infrared spectroscopy (FT-IR) was effective in differentiating genetically distinct strains compared with whole-genome sequencing, but was less effective as a proxy for genetic distance. CONCLUSIONS: Use of a streamlined set of conditions selected for cultivation of skin, oral, and gut microbiota facilitates recovery of dominant microbes and their strain variants from a relatively large sample set. FT-IR spectroscopy allows rapid differentiation of strain variants, but these differences are limited in recapitulating genetic distance. Our data highlights the strength of our cultivation and characterization pipeline, which is in throughput, comparisons with high-resolution genomic data, and rapid identification of strain variation.

Ectopic mineralisation of the mandibular symphysis in ENT1 knockout mice: A model of dystrophic calcification.

Equilibrative nucleoside transporter 1 (ENT1) transfers nucleosides, such as adenosine, across plasma membranes. We reported previously that mice lacking ENT1 (ENT1 -/- ) exhibit progressive ectopic calcification of spinal tissues-a phenotype resembling diffuse idiopathic skeletal hyperostosis (DISH) in humans. Our objective was to investigate potential calcification of orofacial tissues in ENT1 -/- mice. Heads of wild-type mice and ENT1 -/- mice from 3 to 17 months were evaluated using microcomputed tomography (µCT). Some heads were decalcified and processed for histological assessment. Other heads were examined using energy dispersive X-ray spectroscopy and micro X-ray diffraction. Using µCT, ENT1 -/- mice showed extensive radiopaque lesions within the mandibular symphysis, the severity of which increased with advancing age. Histologically, at 6 months these ectopic radiopacities were found to correspond to acellular, amorphous, eosinophilic material, with no evidence of inflammatory cells. Because lesions were localised to the symphysis, we identified early pathological changes at 3 months and observed that lesions initiated specifically within the fibrocartilage pad. Energy-dispersive X-ray spectroscopy of ectopic lesions revealed large amounts of calcium and phosphorous in a molar ratio of ~1.59, and X-ray diffraction profiles matched that of calcium-deficient hydroxyapatite. This is the first characterisation of ectopic calcifications within the mandibular symphysis of ENT1 -/- mice, indicating a role for ENT1 and adenosine metabolism in regulating calcification of fibrocartilaginous tissues. Moreover, these murine lesions resemble areas of dystrophic calcification in the spinal tissues of humans with DISH. Importantly, ectopic calcifications develop in a reproducible temporal pattern within a well-defined anatomical region and, thus, provide a model for determining the cellular and molecular pathways underlying ectopic calcification in DISH and related disorders.

Dystrophic calcification and heterotopic ossification in fibrocartilaginous tissues of the spine in diffuse idiopathic skeletal hyperostosis (DISH).

Diffuse idiopathic skeletal hyperostosis (DISH) is a prevalent noninflammatory spondyloarthropathy characterized by ectopic mineral formation along the anterolateral aspect of the vertebral column, yet little is known about its underlying pathogenesis. Our objective was to evaluate the histopathological features and composition of ectopic mineral within spinal tissues affected by DISH in humans. Thoracic spine segments from six embalmed cadaveric donors (one female and five males; median age 82 years) meeting the radiographic diagnostic criteria for DISH were evaluated using radiological, histological, and physical analyses. Overall, the histological features of ectopic mineralization at individual motion segments were heterogeneous, including regions of heterotopic ossification and dystrophic calcification. Heterotopic ossifications were characterized by woven and lamellar bone, multifocal areas of metaplastic cartilage, and bony bridges along the anterior aspect of the intervertebral disc space. Dystrophic calcifications were characterized by an amorphous appearance, a high content of calcium and phosphorus, an X-ray diffraction pattern matching that of hydroxyapatite, and radiodensities exceeding that of cortical bone. Dystrophic calcifications were found within the anterior longitudinal ligament and annulus fibrosus in motion segments both meeting and not meeting the radiographic criteria for DISH. In summary, our findings indicate that in DISH, ectopic mineral forms along the anterior aspect of the spine by both heterotopic ossification and dystrophic calcification of fibrocartilaginous tissues. Although both types of ectopic mineralization are captured by current radiographic criteria for DISH, dystrophic calcification may reflect a distinct disease process or an early stage in the pathogenesis of DISH.

Loss of P2X7 nucleotide receptor function leads to abnormal fat distribution in mice.

The P2X7 receptor is an ATP-gated cation channel expressed by a number of cell types. We have shown previously that disruption of P2X7 receptor function results in downregulation of osteogenic markers and upregulation of adipogenic markers in calvarial cell cultures. In the present study, we assessed whether loss of P2X7 receptor function results in changes to adipocyte distribution and lipid accumulation in vivo. Male P2X7 loss-of-function (KO) mice exhibited significantly greater body weight and epididymal fat pad mass than wild-type (WT) mice at 9 months of age. Fat pad adipocytes did not differ in size, consistent with adipocyte hyperplasia rather than hypertrophy. Histological examination revealed ectopic lipid accumulation in the form of adipocytes and/or lipid droplets in several non-adipose tissues of older male KO mice (9-12 months of age). Ectopic lipid was observed in kidney, extraorbital lacrimal gland and pancreas, but not in liver, heart or skeletal muscle. Specifically, lacrimal gland and pancreas from 12-month-old male KO mice had greater numbers of adipocytes in perivascular, periductal and acinar regions. As well, lipid droplets accumulated in the renal tubular epithelium and lacrimal acinar cells. Blood plasma analyses revealed diminished total cholesterol levels in 9- and 12-month-old male KO mice compared with WT controls. Interestingly, no differences were observed in female mice. Moreover, there were no significant differences in food consumption between male KO and WT mice. Taken together, these data establish novel in vivo roles for the P2X7 receptor in regulating adipogenesis and lipid metabolism in an age- and sex-dependent manner.

Subcellular elevation of cytosolic free calcium is required for osteoclast migration.

Osteoclasts are multinucleated cells responsible for the resorption of bone and other mineralized tissues during development, physiological remodeling, and pathological bone loss. Osteoclasts have the ability to resorb substrate while concurrently migrating. However, the subcellular processes underlying migration are not well understood. It has been proposed that, in other cell types, cytosolic free Ca(2+) concentration ([Ca(2+) ]i ) regulates cell protrusion as well as retraction. Integration of these distinct events would require precise spatiotemporal patterning of subcellular Ca(2+) . The large size of osteoclasts offers a unique opportunity to monitor patterns of Ca(2+) during cell migration. We used ratiometric imaging to map [Ca(2+) ]i within rat and mouse osteoclasts. Migration was characterized by lamellipodial outgrowth at the leading edge, along with intermittent retraction of the uropod. Migrating osteoclasts displayed elevation of [Ca(2+) ]i in the uropod, that began prior to retraction. Dissipation of this [Ca(2+) ]i gradient by loading osteoclasts with the Ca(2+) chelator BAPTA abolished uropod retraction, on both glass and mineralized substrates. In contrast, elevation of [Ca(2+) ]i using ionomycin initiated prompt uropod retraction. To investigate downstream effectors, we treated cells with calpain inhibitor-1, which impaired uropod retraction. In contrast, lamellipodial outgrowth at the leading edge of osteoclasts was unaffected by any of these interventions, indicating that the signals regulating outgrowth are distinct from those triggering retraction. The large size of mature, multinucleated osteoclasts allowed us to discern a novel spatiotemporal pattern of Ca(2+) involved in cell migration. Whereas localized elevation of Ca(2+) is necessary for uropod retraction, lamellipod outgrowth is independent of Ca(2+) -a heretofore unrecognized degree of specificity underlying the regulation of osteoclast migration.