Imaging of Alkaline Phosphatase Activity in Bone Tissue

The purpose of this study was to develop a paradigm for quantitative molecular imaging of bone cell activity. We hypothesized the feasibility of non-invasive imaging of the osteoblast enzyme alkaline phosphatase (ALP) using a small imaging molecule in combination with 19Flourine magnetic resonance spectroscopic imaging (19FMRSI). 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), a fluorinated ALP substrate that is activatable to a fluorescent hydrolysis product was utilized as a prototype small imaging molecule. The molecular structure of DiFMUP includes two Fluorine atoms adjacent to a phosphate group allowing it and its hydrolysis product to be distinguished using 19Fluorine magnetic resonance spectroscopy (19FMRS) and 19FMRSI. ALP-mediated hydrolysis of DiFMUP was tested on osteoblastic cells and bone tissue, using serial measurements of fluorescence activity. Extracellular activation of DiFMUP on ALP-positive mouse bone precursor cells was observed. Concurringly, DiFMUP was also activated on bone derived from rat tibia. Marked inhibition of the cell and tissue activation of DiFMUP was detected after the addition of the ALP inhibitor levamisole. 19FMRS and 19FMRSI were applied for the non-invasive measurement of DiFMUP hydrolysis. 19FMRS revealed a two-peak spectrum representing DiFMUP with an associated chemical shift for the hydrolysis product. Activation of DiFMUP by ALP yielded a characteristic pharmacokinetic profile, which was quantifiable using non-localized 19FMRS and enabled the development of a pharmacokinetic model of ALP activity. Application of 19FMRSI facilitated anatomically accurate, non-invasive imaging of ALP concentration and activity in rat bone. Thus, 19FMRSI represents a promising approach for the quantitative imaging of bone cell activity during bone formation with potential for both preclinical and clinical applications

A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease

Translational Modulation of Proteins Expressed from Bicistronic Vectors

Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment

Advances and future directions for management of trauma patients with musculoskeletal injuries

Musculoskeletal injuries are the most common reason for operative procedures in severely injured patients and are major determinants of functional outcomes. In this paper, we summarise advances and future directions for management of multiply injured patients with major musculoskeletal trauma. Improved understanding of fracture healing has created new possibilities for management of particularly challenging problems, such as delayed union and non union of fractures and large bone defects. Optimum timing of major orthopaedic interventions is guided by increased knowledge about the immune response after injury. Individual treatment should be guided by trading off the benefits of early definitive skeletal stabilisation, and the potentially life-threatening risks of systemic complications such as fat embolism, acute lung injury, and multiple organ failure. New methods for measurement of fracture healing and function and quality of life outcomes pave the way for landmark trials that will guide the future management of musculoskeletal injuries

Non-invasive imaging of ALP activity in rat tibia cortical bone.

<p>A: RARE ¹H images of the bone sample anatomy including the rat tibia cortical bone core within the glass vial. B and C: ¹⁹FMRSI-derived parametric maps of regional DiFMUP and hydrolysis product concentrations overlaid onto RARE ¹H images of the bone sample (B and C, respectively). D: ¹⁹FMRSI-derived parametric maps of regional ALP concentration and activity overlaid onto RARE ¹H images of the bone sample.</p

ALP-dependent DiFMUP activation on the surface of ALP-positive osteoblastic bone cells and in the presence of rat tibial bone.

<p>A: Histochemistry detected ALP expression as violet-red staining on 7F2 cells as shown in low and high magnification images 1 and 2, respectively. In contrast, no ALP expression was detectable on the MC3T3-E1#4 cells shown also in low (3) and high magnification (4). B: Activation (hydrolysis) of DiFMUP occurred on 7F2 cells (closed squares), but not on ALP-negative MC3T3-E1#4 cells (open triangles). No-cell background (Bkg) measurements are also presented (open circles). C: Following the separation of cells (CE) and medium (M) and a single wash step (W), the vast majority of the hydrolysis product was found in the medium. Mean values and standard deviations are shown (n = 3). D: DiFMUP in physiological solution was activated in a time-dependent fashion in the presence of highly purified rat tibia bone (closed squares). Addition of the ALP inhibitor levamisole reduced DiFMUP activation (open squares). E: DiFMUP in alkaline solution was activated by a single highly purified tibia bone chip (closed squares). Presence of levamisole significantly suppressed the activation (open squares). Mean values and standard deviations are shown (n = 3).</p

Pharmacokinetics of ALP-dependent DiFMUP activation in the presence of rat tibial bone.

<p>A: Representative serial ¹⁹FMR spectra of DiFMUP and its hydrolysis product in the presence of rat bone. Spectral acquisition was initiated 6 minutes after addition of DiFMUP (20–25 mM range) to highly purified rat bone chips and serial spectra were acquired with 10 min temporal resolution. Peak assignments are as described for previously demonstrated spectra including (1) sodium fluoride, (2,4) DiFMUP and (3,5) DiFMUP hydrolysis product. B: The measured and modeled time course kinetics of unbound DiFMUP (x, —) and its hydrolysis product (x, —) for the ¹⁹FMR spectra seen in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022608#pone-0022608-g003" target="_blank">Fig. 3A</a>. The modeled ALP bound DiFMUP (—) is also shown for comparison.</p