Mechanical-Stress-Related Epigenetic Regulation of ZIC1 Transcription Factor in the Etiology of Postmenopausal Osteoporosis.

Mechanical loading exerts a profound influence on bone density and architecture, but the exact mechanism is unknown. Our study shows that expression of the neurological transcriptional factor zinc finger of the cerebellum 1 (ZIC1) is markedly increased in trabecular bone biopsies in the lumbar spine compared with the iliac crest, skeletal sites of high and low mechanical stress, respectively. Human trabecular bone transcriptome analyses revealed a strong association between ZIC1 mRNA levels and gene transcripts characteristically associated with osteoblasts, osteocytes and osteoclasts. This supposition is supported by higher ZIC1 expression in iliac bone biopsies from postmenopausal women with osteoporosis compared with age-matched control subjects, as well as strongly significant inverse correlation between ZIC1 mRNA levels and BMI-adjusted bone mineral density (BMD) (Z-score). ZIC1 promoter methylation was decreased in mechanically loaded vertebral bone compared to unloaded normal iliac bone, and its mRNA levels correlated inversely with ZIC1 promoter methylation, thus linking mechanical stress to epigenetic control of gene expression. The findings were corroborated in cultures of rat osteoblast progenitors and osteoblast-like cells. This study demonstrates for the first time how skeletal epigenetic changes that are affected by mechanical forces give rise to marked alteration in bone cell transcriptional activity and translate to human bone pathophysiology.

Portable, low cost and sensitive cavity enhanced absorption (CEA) detection.

Absorption is a widely used technique for a range of different applications. It has lower sensitivity than many other techniques such as fluorescence which has 100 to 1000 times higher sensitivity than absorption. Optical cavity approaches have been developed where the light passes back and forth, within the sample, between two high reflectivity mirrors to increase the pathlength and sensitivity. These approaches have not yet, however, been widely used for analytical applications and for point-of-care diagnostics. Here we show a portable cavity enhanced absorption (CEA) spectrometer and a low cost point-of-care (POC) reader with CEA detection with mechanical elements fabricated using 3D printing. The CEA spectrometer can be used in both single pass and multi-pass cavity enhanced mode to provide measurements in the visible region that are very sensitive and over a wide dynamic range. The CEA mode was shown for Rhodamine B dye to increase the pathlength 57.8 fold over single pass measurements and an LOD of 7.1 × 10-11 M. The cost of the CEA POC reader was reduced by use of narrow band LEDs, photodiodes and removal of fibre optic coupling and with a 14 fold increase in the pathlength over conventional single pass microplate readers. The CEA POC reader was demonstrated for immunoassay of C-Reactive Protein (CRP), Procalcitonin (PCT) and Interleukin 6 (IL-6), towards a three biomarker panel to aid the diagnosis of sepsis. The CEA POC reader can be integrated with wireless connectivity for cloud based data sharing. We show here the potential for the wider use of optical cavity approaches where there is a need for sensitive absorption measurements and also for low cost point-of-care diagnostics.

Quantitative Computed Tomography (QCT) of the Distal Forearm in Men Using a Spiral Whole-Body CT Scanner - Description of a Method and Reliability Assessment of the QCT Pro Software.

The Mr F study investigates the pathogenesis of low trauma distal forearm fractures in men and includes volumetric bone mineral density (vBMD) measurements at the ultradistal forearm as there are no current data. A standard 64 slice CT scanner was used to determine if it was possible to adapt the existing Mindways quantitative computed tomography Pro software for measuring vBMD values at the hip and spine sites. For calculation of intra- and interobserver reliability 40 forearm scans out of the 300 available were chosen randomly. The images were analyzed using the Slice Pick module and Bone Investigational Toolkit. The 4% length of the radius was chosen by measuring the length of the radius from the scaphoid fossa distally to the radial head. The acquired image then underwent extraction, isolation, rotation, and selection of region of interest in order to generate a report on vBMD. A cross-sectional image was created to allow the generation of data on the cortical and trabecular components separately. Repeat analyses were undertaken by 3 independent observers who were blinded as to whether the image was from a participant with or without fracture. The images were presented in random order at each time point. The following parameters were recorded: cortical cross sectional area, total vBMD, trabecular vBMD, and cortical vBMD (CvBMD). Data were analyzed by calculating intraclass correlation coefficients for intra- and interobserver reliability. The lowest values occurred at the CvBMD with intraobserver reliability of 0.92 (95% confidence interval [CI] of 0.86-0.96) and interobserver reliability of 0.92 (95% CI 0.89-0.96). All other parameters had reliability values between 0.97 and 0.99 with tighter 95% CI than for CvBMD. The method of adapting the Mindways Pro software using a standard CT to produce vBMD and structural data at the ultradistal radius is reliable.

Multi-site performance evaluation and Sigma metrics of 20 assays on the Atellica chemistry and immunoassay analyzers.

Background The Atellica Solution comprises chemistry (CH) and immunoassay (IM) analyzers. Recently, six early adopter clinical laboratories across Europe evaluated the analytical performance of 20 CH and IM assays. To measure analytical performance quality, Sigma metrics were calculated for individual-site and pooled-site results. Methods Precision, detection capability, linearity, and method comparison studies were performed according to Clinical Laboratory Standards Institute protocols. Global Sigma metrics across sites were calculated from pooled data at the medical decision level using total allowable error (TEa) goals from CLIA for CH assays, and TEa goals from RiliBÄK for IM assays; and, the equation: Sigma metrics=%TEa-%bias/%CV. A pooled %CV was calculated by combining the imprecision obtained from individual sites. Bias calculations were performed against the ADVIA Chemistry system or ADVIA Centaur system using Deming regression analysis (Passing-Bablok regression for electrolytes) on the pooled-site data. The 103 individual-site Sigma metric calculations used individual-site imprecision and pooled-bias. Results The limits of blank and detection results agreed with the manufacturer's claims. Most assays were linear across the assay range tested. Pooled Sigma metrics were good or better (>4 Sigma) for 18 of 20 assays; and, acceptable for urea nitrogen (3.1) and sodium (3.9), the latter values attributable to higher imprecision at one of five sites. Conclusions Sigma metrics for data generated across multiple real-world sites evaluating the Atellica Solution demonstrated good or better performance of greater than 4 Sigma for 18 of 20 assays tested. Overall, results verified the manufacturer's claims that methods were fit for use in clinical laboratories.

Vitamin D receptor (VDR) gene polymorphism and osteoporosis risk in White British men.

In this study, VDR gene ApaI (rs7975232), BsmI (rs 1544410) and TaqI (rs731236) genotypes were compared in men with osteoporosis and male controls. Osteoporosis affects around 20% of all men and overall mortality in the first year after hip fracture is significantly higher in men than women, yet the genetic basis of osteoporosis is less well studied in males. This study consisted of White British males; 69 osteoporosis patients and 122 controls. BMDs at the lumbar spine (vertebrae L1-L4) and hip (femur neck) were measured by dual-energy X-ray absorptiometry (DEXA). The VDR gene ApaI, BsmI and TaqI genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and association analysis was carried out at genotype and haplotype level. Our study suggests that TaqI polymorphism CC genotype frequency is lower in controls and further analysis of genotypes and BMD revealed a significant effect of TaqI polymorphism on Lumbar spine BMD. Two haplotypes (GCC and AAT) were associated with increased osteoporosis risk. In conclusion, VDR gene TaqI polymorphism in recessive mode had a significant effect on lumbar spine BMD within our study. Haplotypes GCC and AAT increase the risk of osteoporosis among White British males.

The Influence of DNA Methylation on Bone Cells.

DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain.

Real-time activity bioassay of single osteoclasts using a silicon nanocrystal-impregnated artificial matrix.

The lack of an in vitro real-time osteoclast (OC) activity assay has hampered mechanistic studies of bone resorption. Such an assay is developed, employing a hydroxyapatite matrix impregnated with alkyl-capped silicon nanocrystals, which is capable of monitoring the time-course of resorption by single osteoclasts. Resorption of the matrix by OC releases the nanocrystals, which are internalized by the cell and detected as an increase in OC luminescence. This particular choice of nanocrystals is motivated by their bright pH-independent luminescence, proportional to concentration, and by their rapid uptake without cytotoxicity. In this in vitro assay, OCs are inhibited by calcitonin (CT) and methyl-ß-cyclodextrin (MCD), and stimulated by receptor activator of nuclear factor kappa-B ligand (RANKL) in the expected manner. The kinetics of the assay exhibit a lag phase representing cell attachment and commencement of resorption processes, followed by a growth of cell luminescence intensity, and the whole time-course is satisfactorily described by the logistic equation.

The clinical utility of bone marker measurements in osteoporosis.

Osteoporosis is characterised by low bone mass and structural deterioration of bone tissue, resulting in increased fragility and susceptibility to fracture. Osteoporotic fractures are a significant cause of morbidity and mortality. Direct medical costs from such fractures in the UK are currently estimated at over two billion pounds per year, resulting in a substantial healthcare burden that is expected to rise exponentially due to increasing life expectancy. Currently bone mineral density is the WHO standard for diagnosis of osteoporosis, but poor sensitivity means that potential fractures will be missed if it is used alone. During the past decade considerable progress has been made in the identification and characterisation of specific biomarkers to aid the management of metabolic bone disease. Technological developments have greatly enhanced assay performance producing reliable, rapid, non-invasive cost effective assays with improved sensitivity and specificity. We now have a greater understanding of the need to regulate pre-analytical sample collection to minimise the effects of biological variation. However, bone turnover markers (BTMs) still have limited clinical utility. It is not routinely recommended to use BTMs to select those at risk of fractures, but baseline measurements of resorption markers are useful before commencement of anti-resorptive treatment and can be checked 3-6 months later to monitor response and adherence to treatment. Similarly, formation markers can be used to monitor bone forming agents. BTMs may also be useful when monitoring patients during treatment holidays and aid in the decision as to when therapy should be recommenced. Recent recommendations by the Bone Marker Standards Working Group propose to standardise research and include a specific marker of bone resorption (CTX) and bone formation (P1NP) in all future studies. It is hoped that improved research in turn will lead to optimised markers for the clinical management of osteoporosis and other bone diseases.

Prostate cancer and osteoporosis.

Adenocarcinoma of the prostate is one of the commonest cancers in the world. Due to a combination of earlier detection and better treatments, survival has increased dramatically. Prostate cancer itself is associated with lower bone density and increased fractures. This is compounded by the use of androgen deprivation therapy, which causes dramatic falls in circulating testosterone and estrogen, resulting in rapid falls in bone density, decreased muscle mass, and increased fracture rates. Bisphosphonates have been demonstrated to prevent and reverse this bone loss, but there are no anti-fracture data. Denosumab, a monoclonal antibody to RANKL, has recently been shown to increase bone density and reduce fracture rates. Prostate cancer also commonly metastasizes to bone where it can cause complications such as fracture and pain. Both zoledronic acid and denosumab have been demonstrated to reduce skeletal related events. Comparative studies would suggest that densosumab may have an advantage over zoledronic acid.

Reduced trabecular bone mineral density and thinner cortices in men with distal forearm fractures.

Men with distal forearm fractures have reduced bone density, an increased risk of osteoporosis and of further fractures. The aim of the study was to investigate the structural determinants of these observations using quantitative CT (qCT). Ninety six men with low-trauma distal forearm fracture and 101 age-matched healthy control subjects were recruited. All subjects underwent a quantitative CT on a standard 64-slice whole body CT scanner. These were analysed on Mindways QCT PRO™ Software to generate volumetric and geometric data at the lumbar spine, femoral neck and total hip, ultra-distal and distal 33 % radius. Biochemical investigations, health questionnaires and measurements of bone turnover were made. Men with fracture had significantly lower total and trabecular vBMD at all sites. The greatest percentage reduction was at the ultra-distal radius (13.5 % total and 11.7 % trabecular vBMD). In the fracture group cortical vBMD was significantly higher in the femoral neck (p < 0.001) and maintained at the ultra-distal radius compared with control subjects. However, cortical cross-sectional area (CSA) and thickness were significantly reduced at the femoral neck (p < 0.001 and p = 0.002 respectively) and forearm sites (CSA ultradistal radius p = 0.001, cortical thickness p = 0.002, CSA distal one third radius p = 0.045 and cortical thickness p = 0.005). Cross sectional moment of inertia (CSMI) and section moduli were significantly reduced at the femoral neck (CSMI1 p = 0.002, CSMI2 p = 0.012 and section moduli Z1 p < 0.001, Z2 p = 0.004) and the ultra-distal radius (CSMI1 p = 0.008 and section moduli Z1 p = 0.018, Z2 p = 0.007). In stepwise logistic regression analysis distal forearm fracture showed the strongest association with a model comprising ultra-distal forearm trabecular vBMD (negative), procollagen type I N-terminal propeptide (PINP, positive) and sex hormone binding globulin (SHBG, negative). In conclusion, these observations explain the structural reasons for the increased fracture risk in men with distal forearm fractures.

Zic1 transcription factor in bone: neural developmental protein regulates mechanotransduction in osteocytes.

A transcriptome analysis compared gene expression in human bone biopsy samples taken from lumbar spine and iliac crest, sites that experience high and low levels of mechanical stress, respectively. The analysis revealed that the zinc finger protein of cerebellum (Zic) family member transcription factor Zic1 was the most up-regulated gene in the lumbar spine (202-fold; P<10(-7)) in comparison with the iliac crest. Software analysis of differential gene expression in the biopsy samples identified the ciliary-related proteins PATCH1 and GLI-Kruppel family members Gli1 and Gli3 as part of a potential molecular network associated with Zic1. RT-PCR confirmed the expression of Zic1, Gli1, and Gli3 and other related key signaling mediators in osteoblastic cells and osteocytes in vitro. Zic1 was immunolocalized in the cytosol and nucleus of the murine osteocyte cell line MLO-Y4 and osteoblast-like cells MC3T3-E1 and in primary rat osteoblasts. MLO-Y4 cells subjected to prolonged oscillatory fluid flow showed increased localization of Zic1 in the nucleus with diminished levels in the cytosol, but no such changes were seen in MC3T3-E1 cells. A shear stress-induced increase in T-cell factor/lymphoid enhancer factor transcriptional activity was abolished by Zic1 gene silencing. These results suggest that Zic1, perhaps together with Gli1 and Gli3, may act as a link between mechanosensing and Wnt signaling. We conclude that Zic1, a neural developmental transcription factor, plays an important role in shear flow mechanotransduction in osteocytes.

Biomarkers for Point-of-Care Diagnosis of Sepsis.

Sepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. In 2017, almost 50 million cases of sepsis were recorded worldwide and 11 million sepsis-related deaths were reported. Therefore, sepsis is the focus of intense research to better understand the complexities of sepsis response, particularly the twin underlying concepts of an initial hyper-immune response and a counter-immunological state of immunosuppression triggered by an invading pathogen. Diagnosis of sepsis remains a significant challenge. Prompt diagnosis is essential so that treatment can be instigated as early as possible to ensure the best outcome, as delay in treatment is associated with higher mortality. In order to address this diagnostic problem, use of a panel of biomarkers has been proposed as, due to the complexity of the sepsis response, no single marker is sufficient. This review provides background on the current understanding of sepsis in terms of its epidemiology, the evolution of the definition of sepsis, pathobiology and diagnosis and management. Candidate biomarkers of interest and how current and developing point-of-care testing approaches could be used to measure such biomarkers is discussed.

Alkyl-capped silicon nanocrystals lack cytotoxicity and have enhanced intracellular accumulation in malignant cells via cholesterol-dependent endocytosis.

Nanocrystals of various inorganic materials are being considered for application in the life sciences as fluorescent labels and for such therapeutic applications as drug delivery or targeted cell destruction. The potential applications of the nanoparticles are critically compromised due to the well-documented toxicity and lack of understanding about the mechanisms involved in the intracellular internalization. Here intracellular internalization and toxicity of alkyl-capped silicon nanocrystals in human neoplastic and normal primary cells is reported. The capped nanocrystals lack cytotoxicity, and there is a marked difference in the rate and extent of intracellular accumulation of the nanoparticles between human cancerous and non-cancerous primary cells, the rate and extent being higher in the malignant cells compared to normal human primary cells. The exposure of the cells to the alkyl-capped nanocrystals demonstrates no evidence of in vitro cytotoxicity when assessed by cell morphology, apoptosis, and cell viability assays. The internalization of the nanocrystals by Hela and SW1353 cells is almost completely blocked by the pinocytosis inhibitors filipin, cytochalasin B, and actinomycin D. The internalization process is not associated with any surface change in the nanoparticles, as their luminescence spectrum is unaltered upon transport into the cytosol. The observed dramatic difference in the rate and extent of internalization of the nanocrystals between malignant and non-malignant cells therefore offers potential application in the management of human neoplastic conditions.

Endocytosis and Lack of Cytotoxicity of Alkyl-Capped Silicon Quantum Dots Prepared from Porous Silicon.

Freely-dissolved silicon quantum dots were prepared by thermal hydrosilation of 1-undecene at high-porosity porous silicon under reflux in toluene. This reaction produces a suspension of alkyl-capped silicon quantum dots (alkyl SiQDs) with bright orange luminescence, a core Si nanocrystal diameter of about 2.5 nm and a total particle diameter of about 5 nm. Previous work has shown that these particles are rapidly endocytosed by malignant cell lines and have little or no acute toxicity as judged by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for viability and the Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis. We have extended this work to the CACO-2 cell line, an established model for the human small intestinal mucosa, and demonstrate that neither acute nor chronic (14 days) toxicity is observed as judged by cell morphology, viability, ATP production, ROS production and DNA damage (single cell gel electrophoresis) at doses of 50-200 µ g mL - 1 . Quantitative assessment of the extent of uptake of alkyl SiQDs by CACO-2, HeLa, HepG2, and Huh7 cell lines by flow cytometry showed a wide variation. The liver cell lines (HepG2 and Huh7) were the most active and HeLa and CACO-2 showed comparable activity. Previous work has reported a cholesterol-sensitivity of the endocytosis (HeLa), which suggests a caveolin-mediated pathway. However, gene expression analysis by quantitative real-time polymerase chain reaction (RT-PCR) indicates very low levels of caveolins 1 and 2 in HepG2 and much higher levels in HeLa. The data suggest that the mechanism of endocytosis of the alkyl SiQDs is cell-line dependent.

Time-Effect of Donor and Recipient Characteristics on Graft Survival after Kidney Transplantation.

The kidney allocation system (KAS) is based on quality-based "longevity matching" strategies that provide only a momentary snapshot of expected outcomes at the time of transplantation. The purpose of our study was to define on a continuous timeline the relative and mutual interactions of donor and recipient characteristics on graft survival. Total 39,108 subjects who underwent kidney transplant between October 25, 1999 and January 1, 2007 were identified in the United Network for Organ Sharing dataset. Our primary outcome was graft survival. Time-dependent receiver operating characteristic (ROC) curves and area under time-dependent ROC curve (AUC) were used to compare the predictive ability of the two allocation systems. During the first year after transplantation, both donor and recipient models showed identical relevance. From the first to the sixth years, although the two ROC curves were nearly identical, the donor model outweighed the recipient model. Both models intersected again at the sixth year. From that time onward, the ROC curve for recipient characteristics model predominated over the ROC curve for donor characteristics model. The predictive value of the recipient model (AUC = 0.752) was greater than that of the donor model (AUC = 0.673) We hope that this model will provide additional guidance and risk stratification to further optimize organ allocation based on the dynamic interaction of both donor and recipient characteristics over time.

Changes in bone density and bone turnover in patients with rheumatoid arthritis treated with rituximab, results from an exploratory, prospective study.

Data describing the effect of in vivo B cell depletion on general bone loss in patients with rheumatoid arthritis (RA) are limited. Given the pathogenetic role of B cells in RA, it is tempting to speculate that B cell depletion might have a beneficial effect on bone loss. We prospectively investigated the changes in bone mineral density (BMD), bone turnover, inflammation and disease activity before and after rituximab in 45 RA patients over a 12 month period, 36 patients of whom completed the study and were included in the analysis. There was no significant change in our primary endpoint; lumbar spine BMD after 12 months. However, we found a significant decrease in neck of femur (mean -0.017 g/cm2, 95% CI -0.030, -0.004 p = 0.011) and total femur BMD (mean -0.016 g/cm2, 95% CI -0.025, -0.007 p = 0.001). Additionally, there was a significant increase in procollagen type 1 amino-terminal propeptide (P1NP) and bone specific alkaline phosphatase (BAP); biomarkers of bone formation (median change from baseline to 12 months; P1NP 11.3 µg/L, 95% CI -1.1, 24.8 p = 0.025; BAP 2.5 µg/L, 95% CI 1.2, 3.6 p = 0.002), but no significant change in bone resorption or osteocyte markers. The fall in BMD occurred despite improvement in disease control. Post-menopausal women had the lowest mean lumbar spine, femoral and forearm BMD at baseline and after 12 months, additionally they had a higher level of bone turnover throughout the study. In conclusion, BMD was maintained at the lumbar spine and forearm, but fell at the femur sites. A high prevalence of vitamin D deficiency was observed and these patients had lower BMD and evidence of higher bone turnover.

Hepatic surgery-related hypophosphatemia.

This review describes pathophysiology of post-surgical hypophosphatemia (HP), which has particularly high incidence following liver transplantation. HP remains poorly understood; and there is a lack of universally accepted guidelines for its investigation and management. The pathogenesis of HP following major liver surgery has been hypothesized as being due either to excessive utilization by regenerating liver or increased urinary losses of phosphate. This review provides evidence that excessive urinary loss rather than increased Pi uptake by the liver is the most likely mechanism, and this may be mediated by recently described phosphaturic factors, known as phosphatonins. Until recently blood Pi homeostasis had been explained solely in terms of classical hormones, i.e., vitamin D and PTH. It is however increasingly recognized that phosphatonins may play a critical role in the post-operative HP, but the exact mechanism and candidate phosphaturic factor has not yet been identified. In this review, we have described likely mechanisms and suggest candidate phosphatonins that may mediate urinary Pi loss following liver transplantation. We also discuss the biochemical consequences of cellular Pi depletion, which exposes some gaps in the utilization of established knowledge and therefore in the management of HP. The main aspects of pathophysiology of HP and cellular Pi depletion are presented to provide rational for novel biochemical investigations, which are likely to improve monitoring of HP associated metabolic stress as well as extent of severity of HP, and thereby enhance management of these patients.