Evaluation of Vascular Control Mechanisms Utilizing Video Microscopy of Isolated Resistance Arteries of Rats

This protocol describes the use of in vitro television microscopy to evaluate vascular function in isolated cerebral resistance arteries (and other vessels), and describes techniques for evaluating tissue perfusion using Laser Doppler Flowmetry (LDF) and microvessel density utilizing fluorescently labeled Griffonia simplicifolia (GS1) lectin. Current methods for studying isolated resistance arteries at transmural pressures encountered in vivo and in the absence of parenchymal cell influences provide a critical link between in vivo studies and information gained from molecular reductionist approaches that provide limited insight into integrative responses at the whole animal level. LDF and techniques to selectively identify arterioles and capillaries with fluorescently-labeled GS1 lectin provide practical solutions to enable investigators to extend the knowledge gained from studies of isolated resistance arteries. This paper describes the application of these techniques to gain fundamental knowledge of vascular physiology and pathology in the rat as a general experimental model, and in a variety of specialized genetically engineered designer rat strains that can provide important insight into the influence of specific genes on important vascular phenotypes. Utilizing these valuable experimental approaches in rat strains developed by selective breeding strategies and new technologies for producing gene knockout models in the rat, will expand the rigor of scientific premises developed in knockout mouse models and extend that knowledge to a more relevant animal model, with a well understood physiological background and suitability for physiological studies because of its larger size

Elucidation of a Low Spin Cobalt(II) System in a Distorted Tetrahedral Geometry

We have prepared a series of divalent cobalt(II) complexes supported by the [PhBP_3] ligand ([PhBP_3] = [PhB(CH_2PPh_2)_3]-) to probe certain structural and electronic phenomena that arise from this strong field, anionic tris(phosphine) donor ligand. The solid-state structure of the complex [PhBP_3]CoI (1), accompanied by SQUID, EPR, and optical data, indicates that it is a pseudotetrahedral cobalt(II) species with a doublet ground state the first of its type. To our knowledge, all previous examples of 4-coordinate cobalt(II) complexes with doublet ground states have adopted square planar structure types. Complex 1 provided a useful precursor to the corresponding bromide and chloride complexes, {[PhBP_3]Co(μ-Br)}_2, (2), and {[PhBP_3]Co(μ-Cl)}_2, (3). These complexes were similarly characterized and shown to be dimeric in the solid-state. In solution, however, the monomeric low spin form of 2 and 3 dominates at 25 °C. There is spectroscopic evidence for a temperature-dependent monomer/dimer equilibrium in solution for complex 3. Furthermore, the dimers 2 and 3 did not display appreciable antiferromagnetic coupling that is typical of halide and oxo-bridged copper(II) and cobalt(II) dimers. Rather, the EPR and SQUID data for solid samples of 2 and 3 suggest that they have triplet ground states. Complexes 1, 2, and 3 are extremely oxygen sensitive. Thus, stoichiometric oxidation of 1 by dioxygen produced the 4-coordinate, high spin complex [PhB(CH_2P(O)Ph_2)_2(CH_2PPh_2)]CoI, (4), in which the [PhBP_3] ligand had undergone a 4-electron oxidation. Reaction of 1 with TlOAr (Ar = 2,6-Me_2Ph) afforded an example of a 4-coordinate, high spin complex, [PhBP_3]Co(O-2,6-Me_2Ph) (5), with an intact [PhBP_3] ligand. The latter two complexes were spectroscopically and structurally characterized for comparison to complexes 1, 2, and 3. Our data for these complexes collectively suggest that the [PhBP_3] ligand provides an unusually strong ligand-field to these divalent cobalt complexes that is chemically distinct from typical tris(phosphine) donor ligand sets, and distinct from tridentate borato ligands that have been previously studied. Coupling this strong ligand-field with a pronounced axial distortion away from tetrahedral symmetry, a geometric consequence that is enforced by the [PhBP_3] ligand, provides access to monomeric [PhBP_3]CoX complexes with doublet rather than quartet ground states

Establishments’ use of temporary agency workers: the influence of institutions and establishments’ employment strategies

© 2016 Informa UK Limited, trading as Taylor & Francis Group Comparative institutional analyses have added much to our understanding of HRM in different countries, providing powerful arguments against the need for flexible labour markets to boost economic performance. However, existing research has tended to downplay the possibility that variation within countries may result in a well-protected core of workers that grows ever smaller alongside increasing numbers of precarious workers. We draw on data from the World Economic Forum and the European Company Survey to examine how institutions influence establishments’ use of temporary workers in 29 European countries plus Turkey. We analyse the data using (1) principal components analysis to categorize the countries in our analysis, (2) a two-step cluster analysis to draw up groups of establishments by their use of temporary workers and (3) a multilevel logistic regression to examine how the institutional setting of establishments and key establishment characteristics interact to influence workplaces’ use of temporary workers. We show that institutional characteristics shape the prevalence of temporary workers in the 28 European Union member states plus FYR Macedonia and Turkey; however, institutions are not deterministic and important variation in the use of temporary workers depends upon the interaction between establishment characteristics and the establishment’s business system

Induced Anticlinic Ordering and Nanophase Segregation of Bow-Shaped Molecules in a Smectic Solvent

Recent experiments indicate that doping low concentrations of bent-core molecules into calamitic smectic solvents can induce anticlinic and biaxial smectic phases. We have carried out Monte Carlo (MC) simulations of mixtures of rodlike molecules (hard spherocylinders with length/breadth ratio $L_{\rm rod}/D = 5$) and bow- or banana-shaped molecules (hard spherocylinder dimers with length/breadth ratio $L_{ban}/D = 5$ or 2.5 and opening angle $\psi$) to probe the molecular-scale organization and phase behavior of rod/banana mixtures. We find that a low concentration (3%) of $L_{ban}/D = 5$ dimers induces anticlinic (SmC$_A$) ordering in an untilted smectic (SmA) phase for $100^\circ \le \psi < 150^\circ$. For smaller $\psi$, half of each bow-shaped molecule is nanophase segregated between smectic layers, and the smectic layers are untilted. For $L_{ban}/D = 2.5$, no tilted phases are induced. However, with decreasing $\psi$ we observe a sharp transition from {\sl intralamellar} nanophase segregation (bow-shaped molecules segregated within smectic layers) to {\sl interlamellar} nanophase segregation (bow-shaped molecules concentrated between smectic layers) near $\psi = 130^\circ$. These results demonstrate that purely entropic effects can lead to surprisingly complex behavior in rod/banana mixtures.Comment: 5 pages Revtex, 7 postscript figure

Defective cancellous bone structure and abnormal response to PTH in cortical bone of mice lacking Cx43 cytoplasmic C-terminus domain

Connexin 43 (Cx43) forms gap junction channels and hemichannels that allow the communication among osteocytes, osteoblasts, and osteoclasts. Cx43 carboxy-terminal (CT) domain regulates channel opening and intracellular signaling by acting as a scaffold for structural and signaling proteins. To determine the role of Cx43 CT domain in bone, mice in which one allele of full length Cx43 was replaced by a mutant lacking the CT domain (Cx43(ΔCT/fl)) were studied. Cx43(ΔCT/fl) mice exhibit lower cancellous bone volume but higher cortical thickness than Cx43(fl/fl) controls, indicating that the CT domain is involved in normal cancellous bone gain but opposes cortical bone acquisition. Further, Cx43(ΔCT) is able to exert the functions of full length osteocytic Cx43 on cortical bone geometry and mechanical properties, demonstrating that domains other than the CT are responsible for Cx43 function in cortical bone. In addition, parathyroid hormone (PTH) failed to increase endocortical bone formation or energy to failure, a mechanical property that indicates resistance to fracture, in cortical bone in Cx43(ΔCT) mice with or without osteocytic full length Cx43. On the other hand, bone mass and bone formation markers were increased by the hormone in all mouse models, regardless of whether full length or Cx43(ΔCT) were or not expressed. We conclude that Cx43 CT domain is involved in proper bone acquisition; and that Cx43 expression in osteocytes is dispensable for some but not all PTH anabolic actions

Loss of Nmp4 optimizes osteogenic metabolism and secretion to enhance bone quality

A goal of osteoporosis therapy is to restore lost bone with structurally sound tissue. Mice lacking the transcription factor Nuclear Matrix Protein 4 (Nmp4, Zfp384, Ciz, ZNF384) respond to several classes of osteoporosis drugs with enhanced bone formation compared to wild type (WT) animals. Nmp4-/- mesenchymal stem/progenitor cells (MSPCs) exhibit an accelerated and enhanced mineralization during osteoblast differentiation. To address the mechanisms underlying this hyper-anabolic phenotype, we carried out RNA-sequencing and molecular and cellular analyses of WT and Nmp4-/- MSPCs during osteogenesis to define pathways and mechanisms associated with elevated matrix production. We determined that Nmp4 has a broad impact on the transcriptome during osteogenic differentiation, contributing to the expression of over 5,000 genes. Phenotypic anchoring of transcriptional data was performed for the hypothesis-testing arm through analysis of cell metabolism, protein synthesis and secretion, and bone material properties. Mechanistic studies confirmed that Nmp4-/- MSPCs exhibited an enhanced capacity for glycolytic conversion- a key step in bone anabolism. Nmp4-/- cells showed elevated collagen translation and secretion. Expression of matrix genes that contribute to bone material-level mechanical properties were elevated in Nmp4-/- cells, an observation that was supported by biomechanical testing of bone samples from Nmp4-/- and WT mice. We conclude that loss of Nmp4 increases the magnitude of glycolysis upon the metabolic switch, which fuels the conversion of the osteoblast into a super-secretor of matrix resulting in more bone with improvements in intrinsic quality

Bone cell-independent benefits of raloxifene on the skeleton: A novel mechanism for improving bone material properties

Authors' accepted manuscript. Bone Biology Laboratory http://www.iupui.edu/~bonelab/ Department of Anatomy and Cell Biology Indiana University School of Medicine Department of Biomedical Engineering IUPUIRaloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (-OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle x-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.The authors would like to thank Dr. Paul K. Hansma (Department of Physics, University of California, Santa Barbara), for suggesting the soaking technique and Dr. John Okasinski, Advanced Photon Source, for helping collect the WAXS data. Raloxifene was kindly provided by Eli Lilly (Indianapolis, IN, USA) under a Material Transfer Agreement to D.B.B. Eli Lilly was not involved in the study design, analyses or interpretation of the results. We are grateful to Dr. Susan J. Gunst for sharing dog tissue. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357. This work was supported by NIH grants to D.B.B. and M.R.A

Increased FGF23 protects against detrimental cardio-renal consequences during elevated blood phosphate in CKD

The phosphaturic hormone FGF23 is elevated in chronic kidney disease (CKD). The risk of premature death is substantially higher in the CKD patient population, with cardiovascular disease (CVD) as the leading mortality cause at all stages of CKD. Elevated FGF23 in CKD has been associated with increased odds for all-cause mortality; however, whether FGF23 is associated with positive adaptation in CKD is unknown. To test the role of FGF23 in CKD phenotypes, a late osteoblast/osteocyte conditional flox-Fgf23 mouse (Fgf23fl/fl/Dmp1-Cre+/-) was placed on an adenine-containing diet to induce CKD. Serum analysis showed casein-fed Cre+ mice had significantly higher serum phosphate and blood urea nitrogen (BUN) versus casein diet and Cre- genotype controls. Adenine significantly induced serum intact FGF23 in the Cre- mice over casein-fed mice, whereas Cre+ mice on adenine had 90% reduction in serum intact FGF23 and C-terminal FGF23 as well as bone Fgf23 mRNA. Parathyroid hormone was significantly elevated in mice fed adenine diet regardless of genotype, which significantly enhanced midshaft cortical porosity. Echocardiographs of the adenine-fed Cre+ hearts revealed profound aortic calcification and cardiac hypertrophy versus diet and genotype controls. Thus, these studies demonstrate that increased bone FGF23, although associated with poor outcomes in CKD, is necessary to protect against the cardio-renal consequences of elevated tissue phosphate