Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.     

A model of reversible inhibitors in the gastric H+/K+-ATPase binding site determined by rotational echo double resonance NMR

Several close analogues of the noncovalent H(+)/K(+)-ATPase inhibitor SCH28080 (2-methyl-3-cyanomethyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine) have been screened for activity and examined in the pharmacological site of action by solid-state NMR spectroscopy. TMPIP, the 1,2,3-trimethyl analogue of SCH28080, and variants of TMPIP containing fluorine in the phenylmethoxy ring exhibited IC(50) values for porcine H(+)/K(+)-ATPase inhibition falling in the sub-10 microm range. Deuterium NMR spectra of a (2)H-labeled inhibitor titrated into H(+)/K(+)-ATPase membranes revealed that 80-100% of inhibitor was bound to the protein, and K(+)-competition (2)H NMR experiments confirmed that the inhibitor lay within the active site. The active binding conformation of the pentafluorophenylmethoxy analogue of TMPIP was determined from (13)C-(19)F dipolar coupling measurements using the cross-polarization magic angle spinning NMR method, REDOR. It was found that the inhibitor adopts an energetically favorable extended conformation falling between fully planar and partially bowed extremes. These findings allowed a model to be proposed for the binding of this inhibitor to H(+)/K(+)-ATPase based on the results of independent site-directed mutagenesis studies. In the model, the partially bowed inhibitor interacts with Phe(126) close to the N-terminal membrane spanning helix M1 and residues in the extracellular loop bridging membrane helices M5 and M6 and is flanked by residues in M4.     

Biophysical and biochemical properties of oligodeoxy-nucleotides containing 4'-C- and 5'-C-substituted thymidines

We have previously reported oligodeoxynucleotides (ODNs) containing 4'-C- and 5'-C-substituted thymidines, which demonstrated certain favorable biophysical and biochemical properties. In this communication, the hybridization and nuclease stability data of the ODNs along with their capability to induce RNase H activity are presented.     

Distribution of life cycle stages of Sarcoptes scabiei var wombati and effects of severe mange on common wombats in Victoria

Seven female and three male common wombats (Vombatus ursinus) collected from forested areas of Victoria (Australia) over a 10 mo period, 10 April 1997 to 22 February 1998 had at least 30% of their skin affected by severe hyperkeratotic sarcoptic mange. Mangy wombats were grazing during the day, could be readily approached, were in poor body condition, and lacked subcutaneous fat. The anterolateral surface of the body was most heavily parasitised with Sarcoptes scabiei var wombati followed by the posterolateral surface, the dorsal region between the ears, the ears, ventral abdomen, medial aspect of the legs, axillary and inguinal areas, and the dorsal midline. Larvae were the most prevalent life-cycle stage followed by eggs, nymphs, females, and males. Mite numbers and the severity of clinical signs, namely thickness of scale crust and the degree of alopecia, were correlated and were symmetrical on each side of the body. Fissuring of crust and skin only occurred when scale crust was present. Bacterial infections occurred in three of 10 wombats within lymph nodes or the pleural cavity. Lymphoid depletion did not occur in lymph nodes or spleens and prescapular lymph nodes contained a greater amount of nuclear debris in germinal centres than non-mangy wombats. Seven wombats had fatty change in their livers. Gonads of mature wombats were not active or had minimal activity. Significant histopathological changes were not seen in the gastrointestinal tract, kidney, brain, myocardium, spleen, thyroid, reproductive tract, and gonads. Hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and concentrations of hemoglobin, lymphocytes, calcium, glucose, creatinine, total solids, total protein, albumin determined both colormetrically and electrophoretically, and globulins were significantly lower and concentrations of neutrophils, monocytes, phosphorus, urea, glutamate dehydrogenase, aspartate aminotransferase and creatine kinase were significantly higher in mangy versus captive wombats. Concentrations of erythrocytes, mean corpuscular hemoglobin, leucocytes, band neutrophils, eosinophils, nucleated erythrocytes, sodium, potassium, chloride, total bilirubin, alkaline phosphatase, and gamma glutamyltransferase for mangy wombats were not significantly different from that reported for captive wombats. Hematological and pathological changes in mangy wombats were consistent with anemia, inflammation, and changes seen with starvation.     

Chemolithoautotrophy in the marine,magnetotactic bacterial strains MV-1 and MV-2

Magnetite-producing magnetotactic bacteria collected from the oxic–anoxic transition zone of chemically stratified marine environments characterized by O₂/H₂S inverse double gradients, contained internal S-rich inclusions resembling elemental S globules, suggesting they oxidize reduced S compounds that could support autotrophy. Two strains of marine magnetotactic bacteria, MV-1 and MV-2, isolated from such sites grew in O₂-gradient media with H₂S or thiosulfate (S₂O₃^2–) as electron sources and O₂ as electron acceptor or anaerobically with S₂O₃^2– and N₂O as electron acceptor, with bicarbonate (HCO₃^–)/CO₂ as sole C source. Cells grown with H₂S contained S-rich inclusions. Cells oxidized S₂O₃^2– to sulfate (SO₄^2–). Both strains grew microaerobically with formate. Neither grew microaerobically with tetrathionate (S₄O₆^2–), methanol, or Fe²⁺ as FeS, or siderite (FeCO₃). Growth with S₂O₃^2– and radiolabeled ¹⁴C-HCO₃^– showed that cell C was derived from HCO₃^–/CO₂. Cell-free extracts showed ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. Southern blot analyses indicated the presence of a form II RubisCO (cbbM) but no form I (cbbL) in both strains. cbbM and cbbQ, a putative post-translational activator of RubisCO, were identified in MV-1. MV-1 and MV-2 are thus chemolithoautotrophs that use the Calvin–Benson–Bassham pathway. cbbM was also identified in Magnetospirillum magnetotacticum. Thus, magnetotactic bacteria at the oxic–anoxic transition zone of chemically stratified aquatic environments are important in C cycling and primary productivity.     

Mass-transfer-limited nitrate uptake on a coral reef flat,Warraber Island,Torres Strait,Australia

Previous research has identified a relationship between the rate of dissipation of turbulent kinetic energy, , and the mass-transfer-limited rate of uptake by a surface, herein called the ^1/4 law, and suggests this law may be applicable to nutrient uptake on coral reefs. To test this suggestion, nitrate uptake rate and gravitational potential energy loss have been measured for a section of Warraber Island reef flat, Torres Strait, northern Australia. The reef flat section is 3 km long, with a 3 m tidal range, and on the days measured, subject to 6 m s^–1 tradewinds. The measured nitrate uptake coefficient, S , on two consecutive days during the rising tide was 1.23±0.28 and 1.42±0.52×10^–4 m s^–1. The measured loss of gravitational potential energy across the reef flat, GPE , on the same rising tides over a 178 m section was 208±24 and 161±20 kg m^–1 s^–2. Assuming the GPE is dissipated as turbulent kinetic energy in the water column, and using the ^1/4 law, the mass-transfer-limited nitrate uptake coefficient, S_MTL , on the two days was 1.57±0.03 and 1.45±0.04×10^–4 m s^–1. Nitrate uptake on Warraber Island reef flat is close to the mass-transfer limit, and is determined by oceanographic nitrate concentrations and energy climate.Communicated by B.C. Hatcher     

Finite element analysis of the human ACL subjected to passive anterior tibial loads

In this study, a constitutive law based on a nearly incompressible transversely isotropic hyperelastic potential is proposed to describe the mechanical behaviour of the anterior cruciate ligament (ACL). The constitutive formulation is valid for arbitrary kinematics (finite elasticity) and is thermodynamically admissible. Based on anatomic measurements performed on a human cadaveric knee specimen, a three-dimensional continuum finite element model of the ACL was developed. The numerical model was used to simulate clinical procedures such as the Lachman and drawer tests, which are performed to assess the existence and severity of an ACL injury. Finite element analyses showed that the two procedures have distinct effects on the behaviour of the ACL and provided new insights into the stress distributions. Moreover, good qualitative and quantitative agreement was found between the present study and results obtained experimentally in comparable conditions.     

The influence of cavity shape on the stresses in composite dental restorations: a finite element study

Dental restoration adhering to the cavity exhibits fundamentally different load transfer mechanisms from non-adhering restorations. It is therefore questionable that traditional cavity designs are optimal from a purely mechanical point of view when working with composite materials. Drawing from general engineering experience, it can be hypothesised that smooth, well rounded designs with bevelled margins are superior. A finite element model is used in the present investigation to determine the stress field in four different cavity designs as it develops during the curing of the restoration. The results show that a significant reduction of the stress along the adhesive interface between the tooth and the restoration can be achieved through the use of a rounded cavity shape. They also show that the adoption of bevelled margins leads to a reduction of the stress concentration at this location. These results are confirmed by a set of experimental results published in the literature. It is concluded that adhering restorations will perform better from a mechanical point of view if an appropriate cavity shape is selected.     

Improving the comparative map of porcine chromosome 9 with respect to human chromosomes 1, 7 and 11

Conserved segments have been identified by ZOO-FISH between pig chromosome 9 (SSC9) and human chromosomes 1, 7 and 11. To assist in the identification of positional candidate genes for QTL on SSC9, the comparative map was further developed. Primers were designed from porcine EST sequence homologous to genes in regions of human chromosomes 1, 7 and 11. Porcine ESTs were then physically assigned using the INRA somatic cell hybrid panel (INRASCHP) and the high-resolution radiation hybrid panel (IMpRH). Seventeen genes (PEPP3, RAB7L1, FNBP2, MAPKAPK2, GNAI1, ABCB1, STEAP, AKAP9, CYP51A1, SGCE, ROBO4, SIAT4C, GLUL, CACNA1E, PTGS2, C1orf16 and ETS1) were mapped to SSC9, while GUSB, CPSF4 and THG-1 were assigned to SSC3.