Ex vivo quantification of lanthanum and gadolinium in post-mortem human tibiae with estimated barium and iodine concentrations using K x-ray fluorescence.

OBJECTIVES: Lanthanum (La) and gadolinium (Gd) are known to deposit in bone of exposed populations, namely those who are orally administered lanthanum carbonate (LaC, La2(CO3)3) or are injected with Gd-based contrast agents, respectively. In this work, bone La and Gd concentrations from the environment and diet were measured using x-ray fluorescence in ten post-mortem human tibiae. As a secondary objective, bone barium (Ba) and iodine concentrations were estimated. APPROACH: Two calibration lines were produced for La and Gd and the minimum detection limits (MDLs) of the system were determined using a 180° irradiation-detection geometry. MAIN RESULTS: The MDLs of the system were 0.4 µg La g-1 bone mineral and 0.5 µg Gd g-1 bone mineral. The mean concentrations were -0.02 ± 0.1 µg La g-1 bone mineral and 0.1 ± 0.2 µg Gd g-1 bone mineral in tibiae. The average Ba and iodine concentrations estimated from the experimental La calibration line and Monte-Carlo derived sensitivity factors were determined to be 3.4 ± 0.8 µg Ba g-1 bone mineral and -0.5 ± 0.3 µg iodine g-1 bone mineral. Since it was discovered that four donors previously received an iodine-based contrast agent, the mean concentrations in these donors was 27.8 ± 28.4 µg iodine g-1 bone mineral. SIGNIFICANCE: The XRF system has determined baseline concentrations of these four heavy metals in trace quantities from natural exposure pathways (with the exception of iodine in four donors). This indicates that the system can measure low levels in ex vivo tibiae samples and can potentially be further developed for in vivo studies involving live subjects who are directly exposed to these metals.

Development of national system performance metrics for tissue donation, production, and distribution activity.

Canada's federal, provincial, and territorial governments gave Canadian Blood Services a mandate for organ and tissue donation and transplantation, including system performance, data and analytics. In 2012 Canadian Blood Services facilitated an eye and tissue banking workshop focused on standardized specifications and practices. At the workshop, the Canadian tissue community directed Canadian Blood Services to facilitate the development and implementation of a national data stream and analytics. Prior to this no national data was prospectively collected or collated on tissue donation, production or distribution activity. An eye and tissue data committee was formed with representation from eye and tissue banks in all Canadian jurisdictions. A minimum data set, standardized definitions, a data submission form and a quality assurance process was developed. Training was provided to data personal identified by each eye and tissue bank. Data collection was initiated January 1, 2013; with quarterly data submitted to Canadian Blood Services via excel spreadsheet. Data was submitted by sixteen Canadian eye and tissue banks, located in eight of Canada's thirteen provinces and territories, representing a census of activity. Annual data reports, with trend analysis, are generated and distributed to the tissue community to inform operational strategy and system performance improvement. This report provides an overview of the data process and provides visibility to the Canadian tissue donation, production and distribution activities for 3 years; January 1, 2013 to December 31, 2015.

Next-generation RNA sequencing of archival formalin-fixed paraffin-embedded urothelial bladder cancer.

UNLABELLED: Molecular profiling of individual cancers is key to personalised medicine. While sequencing technologies have required stringent sample collection and handling, recent technical advances offer sequencing from tissues collected in routine practice and tissues already stored in archives. In this paper, we establish methods for whole-transcriptome RNA sequencing (RNA-seq) from formalin-fixed paraffin-embedded tissues. We obtain average RNA-seq reads of >100 million per sample using the Illumina HiSeq2000 platform. We find high concordance with results from matching fresh frozen samples (>0.8 Spearman correlation). For validation, we compared low- and high-grade bladder cancer transcriptomes in 49 tumour samples after transurethral resection of bladder tumour. We found 947 differentially expressed protein-coding genes. While high-grade lesions exhibited distinct intertumour transcriptome heterogeneity, the transcriptome of low-grade tumours was homogeneous. PATIENT SUMMARY: In this report, we show that it is now possible to use universally available bladder cancer samples that have been fixed in formalin to perform high-quality transcriptome analysis. This ability will facilitate the development of transcriptome-wide tests based on gene expression correlated with clinical outcome.

Long-range PCR and next-generation sequencing of BRCA1 and BRCA2 in breast cancer.

Individuals and families carrying mutations in BRCA1 and BRCA2 (BRCA1/2) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients (N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.

Control of vertebrate skeletal mineralization by polyphosphates.

BACKGROUND: Skeletons are formed in a wide variety of shapes, sizes, and compositions of organic and mineral components. Many invertebrate skeletons are constructed from carbonate or silicate minerals, whereas vertebrate skeletons are instead composed of a calcium phosphate mineral known as apatite. No one yet knows why the dynamic vertebrate skeleton, which is continually rebuilt, repaired, and resorbed during growth and normal remodeling, is composed of apatite. Nor is the control of bone and calcifying cartilage mineralization well understood, though it is thought to be associated with phosphate-cleaving proteins. Researchers have assumed that skeletal mineralization is also associated with non-crystalline, calcium- and phosphate-containing electron-dense granules that have been detected in vertebrate skeletal tissue prepared under non-aqueous conditions. Again, however, the role of these granules remains poorly understood. Here, we review bone and growth plate mineralization before showing that polymers of phosphate ions (polyphosphates: (PO(3)(-))(n)) are co-located with mineralizing cartilage and resorbing bone. We propose that the electron-dense granules contain polyphosphates, and explain how these polyphosphates may play an important role in apatite biomineralization. PRINCIPAL FINDINGS/METHODOLOGY: The enzymatic formation (condensation) and destruction (hydrolytic degradation) of polyphosphates offers a simple mechanism for enzymatic control of phosphate accumulation and the relative saturation of apatite. Under circumstances in which apatite mineral formation is undesirable, such as within cartilage tissue or during bone resorption, the production of polyphosphates reduces the free orthophosphate (PO(4)(3-)) concentration while permitting the accumulation of a high total PO(4)(3-) concentration. Sequestering calcium into amorphous calcium polyphosphate complexes can reduce the concentration of free calcium. The resulting reduction of both free PO(4)(3-) and free calcium lowers the relative apatite saturation, preventing formation of apatite crystals. Identified in situ within resorbing bone and mineralizing cartilage by the fluorescent reporter DAPI (4',6-diamidino-2-phenylindole), polyphosphate formation prevents apatite crystal precipitation while accumulating high local concentrations of total calcium and phosphate. When mineralization is required, tissue non-specific alkaline phosphatase, an enzyme associated with skeletal and cartilage mineralization, cleaves orthophosphates from polyphosphates. The hydrolytic degradation of polyphosphates in the calcium-polyphosphate complex increases orthophosphate and calcium concentrations and thereby favors apatite mineral formation. The correlation of alkaline phosphatase with this process may be explained by the destruction of polyphosphates in calcifying cartilage and areas of bone formation. CONCLUSIONS/SIGNIFICANCE: We hypothesize that polyphosphate formation and hydrolytic degradation constitute a simple mechanism for phosphate accumulation and enzymatic control of biological apatite saturation. This enzymatic control of calcified tissue mineralization may have permitted the development of a phosphate-based, mineralized endoskeleton that can be continually remodeled.

Radiation effects and radioprotection in MC3T3-E1 mouse calvarial osteoblastic cells.

BACKGROUND: Little is known about the mechanisms and treatment of radiation-induced inhibition of craniofacial bone growth. In an earlier study, the radioprotector amifostine (WR-2721) administered to rabbits before irradiation radioprotected cultured orbitozygomatic complex periosteal osteoblast-like cells. This study assessed the effects of amifostine and its active metabolite on the radiation survival, function, and phenotype of mouse calvarial osteoblast-like cells in a cell culture model. METHODS: MC3T3-E1 newborn mouse calvarial osteoblast-like cells underwent gamma-radiation (0 to 10 Gy) in the presence or absence of either WR-2721 or WR-1065, its active metabolite (10 to 10 M). The effects of radiation with and without drugs were assessed using endpoints of colony-forming ability, cell viability, alkaline phosphatase activity, and expression of osteoblastic phenotype genes (alkaline phosphatase, collagen type I, osteocalcin, and osteopontin). All experiments were replicated at least in triplicate. RESULTS: Irradiation resulted in a dose-dependent inhibition of clonogenic cell survival. Pretreatment with WR-1065, but not WR-2721, resulted in a significant improvement of osteoblast-like cell survival. Specifically, maximum radioprotection was observed with 10 M WR-1065 at a clinically relevant 2-Gy dose of irradiation. No significant radioprotection was observed at the lower (5 x 10 M) concentration of WR-1065. Furthermore, radiation seemed to suppress the expression of osteoblastic phenotype-related genes in a dose-dependent manner. CONCLUSIONS: This study reveals improved survival after irradiation in osteoblast-like cells treated with WR-1065 in vitro and corroborates previous findings from animal models. Further studies using this agent and similar drugs are important for devising strategies to prevent radiation-induced inhibition of craniofacial bone growth.

Radiation-induced craniofacial bone growth inhibition: in vitro cytoprotection in the rabbit orbitozygomatic complex periosteum-derived cell culture.

BACKGROUND: Radiotherapy for the management of head and neck cancer in pediatric patients results in severe inhibition of craniofacial bone growth. Previously, the infant rabbit orbitozygomatic complex was established as an experimental model. Amifostine, a cytoprotective agent, was found effective in preventing radiation-induced bone growth inhibition. This study was designed to investigate the effects radiation on osteogenic cells from infant rabbit orbitozygomatic complex periostea and to assess the effects of cytoprotection in vitro. METHODS: Infant New Zealand White rabbits (n = 18) were randomized into three groups and received radiation (0, 10, or 15 Gy) to both orbitozygomatic complexes. Cell cultures were developed from orbitozygomatic complex periostea, and cell numbers, proliferation, alkaline phosphatase, and collagen type I expression and mineralization were assessed. Subsequently, rabbits (n = 18) were randomized into three groups to receive either radiation at the effective dose, pretreatment with amifostine (300 mg/kg, intravenously, 20 minutes before irradiation) with the effective radiation dose, or no treatment. Cell cultures were developed and tested for proliferation and alkaline phosphatase expression. RESULTS: Irradiation resulted in a significant inhibition of cell numbers (p < 0.001) and proliferation (p < 0.01) at the 15-Gy dose and no statistically significant changes in alkaline phosphatase activity. Collagen type I expression and mineralization were also significantly reduced at the 15-Gy dose. Pretreatment with amifostine significantly (p < 0.05) enhanced the number of surviving cells. CONCLUSIONS: Amifostine is capable of protecting orbitozygomatic complex periosteum-derived osteogenic cells from the deleterious effects of radiation. This study provides the basis for understanding the cellular mechanisms of radiation-induced craniofacial bone growth inhibition and cytoprotection by amifostine.

An in vitro model of radiation-induced craniofacial bone growth inhibition.

Radiation-induced craniofacial bone growth inhibition is a consequence of therapeutic radiation in the survivors of pediatric head and neck cancer. Previously, the infant rabbit orbitozygomatic complex (OZC) was established as a reliable animal model. The purpose of this study was to develop a cell culture model from the rabbit OZC to study the effects of radiation in the craniofacial skeleton. Infant (7-week-old) New Zealand white rabbits were used in this study. Periostea from both OZC were harvested in sterile conditions, introduced into cell culture by way of sequential digestion, and subcultured at confluence. Cultures were analyzed for cellular proliferation (methylthiazoletetrazolium assay), alkaline phosphatase activity, collagen type I expression, and mineralization. Electron microscopy was performed to reveal the in vitro ultrastructure. Subsequently, rabbits were irradiated with sham or 15 Gy radiation, and cell cultures were developed and analyzed for cell numbers. Cell cultures, grown from OZC periostea, expressed osteoblast-like phenotype, with high alkaline phosphatase activity, collagen type 1 expression, and mineralization in an osteogenic environment. Electron microscopy confirmed the characteristic ultrastructural features of osteogenesis in vitro. Finally, significantly (P < 0.01) fewer cells were obtained from animals treated with 15 Gy radiation compared with those from control animals.A primary cell culture with osteoblast-like cellular phenotype was developed from infant rabbit OZC periosteum. This cell culture system responded to in vivo administered radiation by a significant decrease in cell numbers. This in vitro model will be subsequently used to study the cellular mechanisms of radiation and radioprotection in craniofacial osteoblast-like cells.

Metabolic homeostasis and tissue renewal are dependent on beta1,6GlcNAc-branched N-glycans.

Golgi beta1,6-N-acetylglucosaminyltransferase V (Mgat5) produces beta1,6GlcNAc-branched N-glycans on glycoproteins, which increases their affinity for galectins and opposes loss from the cell surface to constitutive endocytosis. Oncogenic transformation increases Mgat5 expression, increases beta1,6GlcNAc-branched N-glycans on epidermal growth factor and transforming growth factor-beta receptors, and enhances sensitivities to ligands, cell motility, and tumor metastasis. Here, we demonstrate that Mgat5(-/-) mouse embryonic fibroblasts (MEFs) display reduced sensitivity to anabolic cytokines and reduced glucose uptake and proliferation. Mgat5(-/-) mice are also hypoglycemic, resistant to weight gain on a calorie-enriched diet, hypersensitive to fasting, and display increased oxidative respiration and reduced fecundity. Serum-dependent activation of the extracellular response kinase (growth) and Smad2/3 (arrest) pathways in Mgat5(-/-) MEFs and bone marrow cells reveals an imbalance favoring arrest. Mgat5(-/-) mice have fewer muscle satellite cells, less osteogenic activity in bone marrow, and accelerated loss of muscle and bone mass with aging. Our results suggest that beta1,6GlcNAc-branched N-glycans promote sensitivity to anabolic cytokines, and increase fat stores, tissue renewal, and longevity.

alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling.

Soluble transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP)-binding proteins are widely distributed in mammalian tissues and control cytokine access to membrane signaling receptors. The serum and bone-resident glycoprotein alpha2-HS-glycoprotein/fetuin (ASHG) binds to TGF-beta/BMP cytokines and blocks TGF-beta1 binding to cell surface receptors. Therefore, we examined bone growth and remodeling phenotypes in ASHG-deficient mice. The skeletal structure of Ahsg(-/-) mice appeared normal at birth, but abnormalities were observed in adult Ahsg(-/-) mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age in Ahsg(-/-) mice. However, bone formation was increased in Ahsg(-/-) mice as indicated by greater cortical thickness, accelerated trabecular bone remodeling, and increased osteoblast numbers on bone surfaces. The normal age-related increase in cortical thickness and bone mineral density was accelerated in Ahsg(-/-) mice and was associated with increased energy required to fracture. Bone formation in response to implanted BMP cytokine extended further from the implant in Ahsg(-/-) compared with Ahsg(+/+) mice, confirming the interaction between ASHG and TGF-beta/BMP cytokines in vivo. Our results demonstrate that ASHG blocks TGF-beta-dependent signaling in osteoblastic cells, and mice lacking ASHG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis.