Osteogenic preconditioning in perfusion bioreactors improves vascularization and bone formation by human bone marrow aspirates.

Cell-derived extracellular matrix (ECM) provides a niche to promote osteogenic differentiation, cell adhesion, survival, and trophic factor secretion. To determine whether osteogenic preconditioning would improve the bone-forming potential of unfractionated bone marrow aspirate (BMA), we perfused cells on ECM-coated scaffolds to generate naïve and preconditioned constructs, respectively. The composition of cells selected from BMA was distinct on each scaffold. Naïve constructs exhibited robust proangiogenic potential in vitro, while preconditioned scaffolds contained more mesenchymal stem/stromal cells (MSCs) and endothelial cells (ECs) and exhibited an osteogenic phenotype. Upon implantation into an orthotopic calvarial defect, BMA-derived ECs were present in vessels in preconditioned implants, resulting in robust perfusion and greater vessel density over the first 14 days compared to naïve implants. After 10 weeks, human ECs and differentiated MSCs were detected in de novo tissues derived from naïve and preconditioned scaffolds. These results demonstrate that bioreactor-based preconditioning augments the bone-forming potential of BMA

Mitochondrial dysfunction, a probable cause of persistent oxidative stress after exposure to ionizing radiation

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability

Nitric Oxide Synthase Inhibition Enhances the Antitumor Effect of Radiation in the Treatment of Squamous Carcinoma Xenografts

This study tests whether the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA), combines favorably with ionizing radiation (IR) in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01). Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control

Mitochondria-dependent signalling pathway are involved in the early process of radiation-induced bystander effects

Bystander effects induced by cytoplasmic irradiation have been reported recently. However, the mechanism(s) underlying, such as the functional role of mitochondria, is not clear. In the present study, we used either mtDNA-depleted (ρ0) AL or normal (ρ+) AL cells as irradiated donor cells and normal human skin fibroblasts as receptor cells in a series of medium transfer experiments to investigate the mitochondria-related signal process. Our results indicated that mtDNA-depleted cells or normal AL cells treated with mitochondrial respiratory chain function inhibitors had an attenuated γ-H2AX induction, which indicates that mitochondria play a functional role in bystander effects. Moreover, it was found that treatment of normal AL donor cells with specific inhibitors of NOS, or inhibitor of mitochondrial calcium uptake (ruthenium red) significantly decreased γ-H2AX induction and that radiation could stimulate cellular NO and O2•− production in irradiated ρ+ AL cells, but not in ρ0 AL cells. These observations, together with the findings that ruthenium red treatment significantly reduced the NO and O2•− levels in irradiated ρ+ AL cells, suggest that radiation-induced NO derived from mitochondria might be an intracellular bystander factor and calcium-dependent mitochondrial NOS might play an essential role in the process

Evaluation of the current knowledge limitations in breast cancer research: a gap analysis

BACKGROUND A gap analysis was conducted to determine which areas of breast cancer research, if targeted by researchers and funding bodies, could produce the greatest impact on patients. METHODS Fifty-six Breast Cancer Campaign grant holders and prominent UK breast cancer researchers participated in a gap analysis of current breast cancer research. Before, during and following the meeting, groups in seven key research areas participated in cycles of presentation, literature review and discussion. Summary papers were prepared by each group and collated into this position paper highlighting the research gaps, with recommendations for action. RESULTS Gaps were identified in all seven themes. General barriers to progress were lack of financial and practical resources, and poor collaboration between disciplines. Critical gaps in each theme included: (1) genetics (knowledge of genetic changes, their effects and interactions); (2) initiation of breast cancer (how developmental signalling pathways cause ductal elongation and branching at the cellular level and influence stem cell dynamics, and how their disruption initiates tumour formation); (3) progression of breast cancer (deciphering the intracellular and extracellular regulators of early progression, tumour growth, angiogenesis and metastasis); (4) therapies and targets (understanding who develops advanced disease); (5) disease markers (incorporating intelligent trial design into all studies to ensure new treatments are tested in patient groups stratified using biomarkers); (6) prevention (strategies to prevent oestrogen-receptor negative tumours and the long-term effects of chemoprevention for oestrogen-receptor positive tumours); (7) psychosocial aspects of cancer (the use of appropriate psychosocial interventions, and the personal impact of all stages of the disease among patients from a range of ethnic and demographic backgrounds). CONCLUSION Through recommendations to address these gaps with future research, the long-term benefits to patients will include: better estimation of risk in families with breast cancer and strategies to reduce risk; better prediction of drug response and patient prognosis; improved tailoring of treatments to patient subgroups and development of new therapeutic approaches; earlier initiation of treatment; more effective use of resources for screening populations; and an enhanced experience for people with or at risk of breast cancer and their families. The challenge to funding bodies and researchers in all disciplines is to focus on these gaps and to drive advances in knowledge into improvements in patient care

Differentiation-Dependent Secretion of Proangiogenic Factors by Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a promising cell population for cell-based bone repair due to their proliferative potential, ability to differentiate into bone-forming osteoblasts, and their secretion of potent trophic factors that stimulate angiogenesis and neovascularization. To promote bone healing, autogenous or allogeneic MSCs are transplanted into bone defects after differentiation to varying degrees down the osteogenic lineage. However, the contribution of the stage of osteogenic differentiation upon angiogenic factor secretion is unclear. We hypothesized that the proangiogenic potential of MSCs was dependent upon their stage of osteogenic differentiation. After 7 days of culture, we observed the greatest osteogenic differentiation of MSCs when cells were cultured with dexamethasone (OM+). Conversely, VEGF protein secretion and upregulation of angiogenic genes were greatest in MSCs cultured in growth media (GM). Using conditioned media from MSCs in each culture condition, GM-conditioned media maximized proliferation and enhanced chemotactic migration and tubule formation of endothelial colony forming cells (ECFCs). The addition of a neutralizing VEGF165/121 antibody to conditioned media attenuated ECFC proliferation and chemotactic migration. ECFCs seeded on microcarrier beads and co-cultured with MSCs previously cultured in GM in a fibrin gel exhibited superior sprouting compared to MSCs previously cultured in OM+. These results confirm that MSCs induced farther down the osteogenic lineage possess reduced proangiogenic potential, thereby providing important findings for consideration when using MSCs for bone repair

Resolution of Joint Molecules by RuvABC and RecG Following Cleavage of the Escherichia coli Chromosome by EcoKI

DNA double-strand breaks can be repaired by homologous recombination involving the formation and resolution of Holliday junctions. In Escherichia coli, the RuvABC resolvasome and the RecG branch-migration enzyme have been proposed to act in alternative pathways for the resolution of Holliday junctions. Here, we have studied the requirements for RuvABC and RecG in DNA double-strand break repair after cleavage of the E. coli chromosome by the EcoKI restriction enzyme. We show an asymmetry in the ability of RuvABC and RecG to deal with joint molecules in vivo. We detect linear DNA products compatible with the cleavage-ligation of Holliday junctions by the RuvABC pathway but not by the RecG pathway. Nevertheless we show that the XerCD-mediated pathway of chromosome dimer resolution is required for survival regardless of whether the RuvABC or the RecG pathway is active, suggesting that crossing-over is a common outcome irrespective of the pathway utilised. This poses a problem. How can cells resolve joint molecules, such as Holliday junctions, to generate crossover products without cleavage-ligation? We suggest that the mechanism of bacterial DNA replication provides an answer to this question and that RecG can facilitate replication through Holliday junctions

Isospin symmetry in B(E2) values: Coulomb excitation study of Mg-21

The $T_z$~=~$-\frac{3}{2}$ nucleus ${}^{21}$Mg has been studied by Coulomb excitation on ${}^{196}$Pt and ${}^{110}$Pd targets. A 205.6(1)-keV $\gamma$-ray transition resulting from the Coulomb excitation of the $\frac{5}{2}^+$ ground state to the first excited $\frac{1}{2}^+$ state in ${}^{21}$Mg was observed for the first time. Coulomb excitation cross-section measurements with both targets and a measurement of the half-life of the $\frac{1}{2}^+$ state yield an adopted value of $B(E2;\frac{5}{2}^+\rightarrow\frac{1}{2}^+)$~=~13.3(4)~W.u. A new excited state at 1672(1)~keV with tentative $\frac{9}{2}^+$ assignment was also identified in ${}^{21}$Mg. This work demonstrates large difference of the $B(E2;\frac{5}{2}^+\rightarrow\frac{1}{2}^+)$ values between $T$~=~$\frac{3}{2}$, $A$~=~21 mirror nuclei. The difference is investigated in the shell-model framework employing both isospin conserving and breaking USD interactions and using modern \textsl{ab initio} nuclear structure calculations, which have recently become applicable in the $sd$ shell.Comment: 8 pages, 6 figures, submitted to Phys. Rev. C, Rapid Communicatio

Healthcare professionals’ views on patient-centered care in hospitals

Background: Patient-centered care (PCC) is a main determinant of care quality. Research has shown that PCC is a multi-dimensional concept, and organizations that provide PCC well report better patient and organizational outcomes. However, little is known about the relative importance of PCC dimensions. The aim of this study was therefore to investigate the relative importance of the eight dimensions of PCC according to hospital-based healthcare professionals, and examine whether their viewpoints are determined by context. Methods: Thirty-four healthcare professionals (16 from the geriatrics department, 15 from a surgical intensive care unit, 3 quality employees) working at a large teaching hospital in New York City were interviewed using Q methodology. Participants were asked to rank 35 statements representing eight dimensions of PCC extracted from the literature: patient preferences, physical comfort, coordination of care, emotional support, acce

A Robust Statistical Method for Association-Based eQTL Analysis

Background: It has been well established that theoretical kernel for recently surging genome-wide association study (GWAS) is statistical inference of linkage disequilibrium (LD) between a tested genetic marker and a putative locus affecting a disease trait. However, LD analysis is vulnerable to several confounding factors of which population stratification is the most prominent. Whilst many methods have been proposed to correct for the influence either through predicting the structure parameters or correcting inflation in the test statistic due to the stratification, these may not be feasible or may impose further statistical problems in practical implementation. Methodology: We propose here a novel statistical method to control spurious LD in GWAS from population structure by incorporating a control marker into testing for significance of genetic association of a polymorphic marker with phenotypic variation of a complex trait. The method avoids the need of structure prediction which may be infeasible or inadequate in practice and accounts properly for a varying effect of population stratification on different regions of the genome under study. Utility and statistical properties of the new method were tested through an intensive computer simulation study and an association-based genome-wide mapping of expression quantitative trait loci in genetically divergent human populations. Results/Conclusions: The analyses show that the new method confers an improved statistical power for detecting genuin