Characterising nitric oxide-mediated metabolic benefits of low-dose ultraviolet radiation in the mouse: a focus on brown adipose tissue.

AIMS/HYPOTHESIS: Exposure to sunlight has the potential to suppress metabolic dysfunction and obesity. We previously demonstrated that regular exposure to low-doses of ultraviolet radiation (UVR) reduced weight gain and signs of diabetes in male mice fed a high-fat diet, in part via release of nitric oxide from skin. Here, we explore further mechanistic pathways through which low-dose UVR exerts these beneficial effects. METHODS: We fed mice with a luciferase-tagged Ucp1 gene (which encodes uncoupling protein-1 [UCP-1]), referred to here as the Ucp1 luciferase transgenic mouse ('Thermomouse') a high-fat diet and examined the effects of repeated exposure to low-dose UVR on weight gain and development of metabolic dysfunction as well as UCP-1-dependent thermogenesis in interscapular brown adipose tissue (iBAT). RESULTS: Repeated exposure to low-dose UVR suppressed the development of glucose intolerance and hepatic lipid accumulation via dermal release of nitric oxide while also reducing circulating IL-6 (compared with mice fed a high-fat diet only). Dietary nitrate supplementation did not mimic the effects of low-dose UVR. A single low dose of UVR increased UCP-1 expression (by more than twofold) in iBAT of mice fed a low-fat diet, 24 h after exposure. However, in mice fed a high-fat diet, there was no effect of UVR on UCP-1 expression in iBAT (compared with mock-treated mice) when measured at regular intervals over 12 weeks. More extensive circadian studies did not identify any substantial shifts in UCP-1 expression in mice exposed to low-dose UVR, although skin temperature at the interscapular site was reduced in UVR-exposed mice. The appearance of cells with a white adipocyte phenotype ('whitening') in iBAT induced by consuming the high-fat diet was suppressed by exposure to low-dose UVR in a nitric oxide-dependent fashion. Significant shifts in the expression of important core gene regulators of BAT function (Dio2, increased more than twofold), fatty acid transport (increased Fatp2 [also known as Slc27a2]), lipolysis (decreased Atgl [also known as Pnpla2]), lipogenesis (decreased Fasn) and inflammation (decreased Tnf), and proportions of macrophages (increased twofold) were observed in iBAT of mice exposed to low-dose UVR. These effects were independent of nitric oxide released from skin. CONCLUSIONS/INTERPRETATION: Our results suggest that non-burning (low-dose) UVR suppresses the BAT 'whitening', steatotic and pro-diabetic effects of consuming a high-fat diet through skin release of nitric oxide, with some metabolic and immune pathways in iBAT regulated by UVR independently of nitric oxide.

Choline kinase ß mutant mice exhibit reduced phosphocholine, elevated osteoclast activity, and low bone mass.

The maintenance of bone homeostasis requires tight coupling between bone-forming osteoblasts and bone-resorbing osteoclasts. However, the precise molecular mechanism(s) underlying the differentiation and activities of these specialized cells are still largely unknown. Here, we identify choline kinase ß (CHKB), a kinase involved in the biosynthesis of phosphatidylcholine, as a novel regulator of bone homeostasis. Choline kinase ß mutant mice (flp/flp) exhibit a systemic low bone mass phenotype. Consistently, osteoclast numbers and activity are elevated in flp/flp mice. Interestingly, osteoclasts derived from flp/flp mice exhibit reduced sensitivity to excessive levels of extracellular calcium, which could account for the increased bone resorption. Conversely, supplementation of cytidine 5'-diphosphocholine in vivo and in vitro, a regimen that bypasses CHKB deficiency, restores osteoclast numbers to physiological levels. Finally, we demonstrate that, in addition to modulating osteoclast formation and function, loss of CHKB corresponds with a reduction in bone formation by osteoblasts. Taken together, these data posit CHKB as a new modulator of bone homeostasis.

Can micro-computed tomography imaging improve interpretation of macroscopic margin assessment of specimen radiography in excised breast specimens?

INTRODUCTION: Peri-operative macroscopic margin assessment with standard intraoperative specimen radiography (IOSR) results in improved re-excision rates in excised breast tissue specimens but is limited. This study sought to improve the intraoperative margin assessment on standard IOSR techniques by utilizing noninvasive X-ray micro-computed tomography (micro-CT) imaging of breast tissue specimens to compare margins in three-dimensional with two-dimensional IOSR. METHODS: Patients with impalpable breast carcinoma, or suspected breast carcinoma, who were eligible for breast-conserving surgery were recruited. Margins were assessed within each specimen using standard IOSR, micro-CT, and histology techniques. RESULTS: Six malignant and three benign lesions were included for the analysis in this study. Micro-CT identified the same positive margin as IOSR in 3 out of 6 malignancies. However, margin status identified by micro-CT was concordant with pathological assessment in only one specimen. In comparison, margin assessment by IOSR correctly correlated with pathological margin status in three malignant specimens. CONCLUSION: The use of micro-CT imaging in this study did not improve margin assessment in impalpable breast specimens when compared to standard specimen radiography (SR) assessment. However, future improvements in sample preparation and CT image acquisition processes may enhance the potential of micro-CT as a valuable imaging tool for improving margin assessment.

Low-dose UV radiation before running wheel access activates brown adipose tissue.

In previous preclinical studies, low (non-burning) doses of UV radiation (UVR) limited weight gain and metabolic dysfunction in mice fed with a high-fat diet. Here, we explored the effects of low-dose UVR on physical activity and food intake and mechanistic pathways in interscapular brown adipose tissue (iBAT). Young adult C57Bl/6J male mice, housed as individuals, were fed a high-fat diet and exposed to low-dose UVR (sub-oedemal, 1 kJ/m2 UVB, twice-a-week) or 'mock' treatment, with or without running wheel access (2 h, for 'moderate' physical activity) immediately after phototherapy. There was no difference in distance run in mice exposed to UVR or mock-treated over 12 weeks of exposure to running wheels (P = 0.14). UVR (alone) did not significantly affect food intake, adiposity, or signs of glucose dysfunction. Access to running wheels increased food intake (after 10 weeks, P ≤ 0.02) and reduced gonadal white adipose tissue and iBAT mass (P ≤ 0.03). Body weight and hepatic steatosis were lowest in mice exposed to UVR with running wheel access. In the iBAT of mice exposed to UVR and running wheels, elevated Atgl, Cd36, Fasn, Igf1, Pparγ, and Ucp1 mRNAs and reduced CD11c on F4-80 + MHC class II+ macrophages were observed, while renal Sglt2 mRNA levels were increased, compared to high-fat diet alone (P ≤ 0.03). Blood levels of 25-hydroxyvitamin D were not increased by exposure to UVR and/or access to running wheels. In conclusion, when combined with physical activity, low-dose UVR may more effectively limit adiposity (specifically, body weight and hepatic steatosis) and modulate metabolic and immune pathways in iBAT.

Commissioning and performance characteristics of a pre-clinical image-guided radiotherapy system.

Characteristics of a small-animal radiotherapy device, the X-RAD SmART, are described following commissioning of the device for pre-clinical radiotherapy research. Performance characteristics were assessed using published standards and compared with previous results published for similar systems. Operational radiation safety was established. Device X-ray beam quality and output dose-rate were found to be consistent with those reported for similar devices. Output steadily declined over 18 months though remained within tolerance levels. There is considerable variation in output factor across the international installations for the smallest field size (varying by more than 30% for 2.5 mm diameter fields). Measured depth dose and profile data was mostly consistent with that published, with some differences in penumbrae and generally reduced flatness. Target localisation is achieved with an imaging panel and with automatic corrections for panel flex and device mechanical instability, targeting within 0.2 mm is achievable. The small-animal image-guided radiotherapy platform has been implemented and assessed and found to perform as specified. The combination of kV energy and high spatial precision make it suitable for replicating clinical dose distributions at the small-animal scale, though dosimetric uncertainties for the narrowest fields need to be acknowledged.

Optimisation of multiplex immunofluorescence for a non-spectral fluorescence scanning system.

The use of multi-colour immunofluorescence (IF) for immunophenotyping in formalin-fixed paraffin-embedded tissue sections is gaining popularity worldwide. This technique allows for the simultaneous detection of multiple markers on the same tissue section, thereby yielding more complex information than is possible by chromogenic immunohistochemistry (IHC). However, many commercially-available multiplex IF kits are designed for use in conjunction with a multispectral imaging system, to which many research groups have limited access. Here we present two 5-colour IF panels designed for T cell characterisation in human colorectal tissue, which can be imaged using a non-spectral fluorescence slide scanner with standard band-pass filters. We describe the optimisation process and the key considerations in developing a multiplex fluorescence assay, and discuss some of the advantages and disadvantages of using multiplex IF with a non-spectral imaging system.

Loss of protein kinase C-δ protects against LPS-induced osteolysis owing to an intrinsic defect in osteoclastic bone resorption.

Bone remodeling is intrinsically regulated by cell signaling molecules. The Protein Kinase C (PKC) family of serine/threonine kinases is involved in multiple signaling pathways including cell proliferation, differentiation, apoptosis and osteoclast biology. However, the precise involvement of individual PKC isoforms in the regulation of osteoclast formation and bone homeostasis remains unclear. Here, we identify PKC-δ as the major PKC isoform expressed among all PKCs in osteoclasts; including classical PKCs (-α, -ß and -γ), novel PKCs (-δ, -ε, -η and -θ) and atypical PKCs (-ι/λ and -ζ). Interestingly, pharmacological inhibition and genetic ablation of PKC-δ impairs osteoclastic bone resorption in vitro. Moreover, disruption of PKC-δ activity protects against LPS-induced osteolysis in mice, with osteoclasts accumulating on the bone surface failing to resorb bone. Treatment with the PKC-δ inhibitor Rottlerin, blocks LPS-induced bone resorption in mice. Consistently, PKC-δ deficient mice exhibit increased trabeculae bone containing residual cartilage matrix, indicative of an osteoclast-rich osteopetrosis phenotype. Cultured ex vivo osteoclasts derived from PKC-δ null mice exhibit decreased CTX-1 levels and MARKS phosphorylation, with enhanced formation rates. This is accompanied by elevated gene expression levels of cathepsin K and PKC -α, -γ and -ε, as well as altered signaling of pERK and pcSrc416/527 upon RANKL-induction, possibly to compensate for the defects in bone resorption. Collectively, our data indicate that PKC-δ is an intrinsic regulator of osteoclast formation and bone resorption and thus is a potential therapeutic target for pathological osteolysis.

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering.

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 µm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.