Receptor activator of NF-kB (RANK) expression in primary tumors associates with bone metastasis occurrence in breast cancer patients

Background

Receptor activator of NFkB (RANK), its ligand (RANKL) and the decoy receptor of RANKL (osteoprotegerin, OPG) play a pivotal role in bone remodeling by regulating osteoclasts formation and activity. RANKL stimulates migration of RANK-expressing tumor cells in vitro, conversely inhibited by OPG.

Materials and Methods

We examined mRNA expression levels of RANKL/RANK/OPG in a publicly available microarray dataset of 295 primary breast cancer patients. We next analyzed RANK expression by immunohistochemistry in an independent series of 93 primary breast cancer specimens and investigated a possible association with clinicopathological parameters, bone recurrence and survival.

Results

Microarray analysis showed that lower RANK and high OPG mRNA levels correlate with longer overall survival (P = 0.0078 and 0.0335, respectively) and disease-free survival (P = 0.059 and 0.0402, respectively). Immunohistochemical analysis of RANK showed a positive correlation with the development of bone metastases (P = 0.023) and a shorter skeletal disease-free survival (SDFS, P = 0.037). Specifically, univariate analysis of survival showed that “RANK-negative” and “RANK-positive” patients had a SDFS of 105.7 months (95% CI: 73.9–124.4) and 58.9 months (95% CI: 34.7–68.5), respectively. RANK protein expression was also associated with accelerated bone metastasis formation in a multivariate analysis (P = 0.029).

Conclusions

This is the first demonstration of the role of RANK expression in primary tumors as a predictive marker of bone metastasis occurrence and SDFS in a large population of breast cancer patients.     

Fission yeast MO25 protein is localized at SPB and septum and is essential for cell morphogenesis

Cell morphogenesis is of fundamental significance in all eukaryotes for development, differentiation, and cell proliferation. In fission yeast, Drosophila Furry-like Mor2 plays an essential role in cell morphogenesis in concert with the NDR/Tricornered kinase Orb6. Mutations of these genes result in the loss of cell polarity. Here we show that the conserved proteins, MO25-like Pmo25, GC kinase Nak1, Mor2, and Orb6, constitute a morphogenesis network that is important for polarity control and cell separation. Intriguingly, Pmo25 was localized at the mitotic spindle pole bodies (SPBs) and then underwent translocation to the dividing medial region upon cytokinesis. Pmo25 formed a complex with Nak1 and was required for both the localization and kinase activity of Nak1. Pmo25 and Nak1 in turn were essential for Orb6 kinase activity. Further, the Pmo25 localization at the SPBs and the Nak1-Orb6 kinase activities during interphase were under the control of the Cdc7 and Sid1 kinases in the septation initiation network (SIN), suggesting a functional linkage between SIN and the network for cell morphogenesis/separation following cytokinesis.     

Automatic generation of accurate subject-specific bone finite element models to be used in clinical studies

Most of the finite element models of bones used in orthopaedic biomechanics research are based on generic anatomies. However, in many cases it would be useful to generate from CT data a separate finite element model for each subject of a study group. In a recent study a hexahedral mesh generator based on a grid projection algorithm was found very effective in terms of accuracy and automation. However, so far the use of this method has been documented only on data collected in vitro and only for long bones. The present study was aimed at verifying if this method represents a procedure for the generation of finite element models of human bones from data collected in vivo, robust, accurate, automatic and general enough to be used in clinical studies. Robustness, automation and numerical accuracy of the proposed method were assessed on five femoral CT data sets of patients affected by various pathologies. The generality of the method was verified by processing a femur, an ileum, a phalanx, a proximal femur reconstruction, and the micro-CT of a small sample of spongy bone. The method was found robust enough to cope with the variability of the five femurs, producing meshes with a numerical accuracy and a computational weight comparable to those found in vitro. Even when the method was used to process the other bones the levels of mesh conditioning remained within acceptable limits. Thus, it may be concluded that the method presents a generality sufficient to cope with almost any orthopaedic application.     

Molecular evolution of haemoglobins of polar fishes

The Arctic and the Antarctic differ by age and isolation of the respective marine faunas. Antarctic fish are highly stenothermal, in response to stable water temperatures, whereas the Arctic ones are exposed to seasonal and latitudinal temperature variations. The knowledge of the mechanisms of phenotypic response to cold exposure in species of both polar habitats offers fundamental insights into the nature of environmental adaptation. In the process of cold adaptation, the evolutionary trend of Antarctic fish has led to unique specialisations, including modification of haematological characteristics, e.g. decreased amounts and multiplicity of haemoglobins.Unlike Antarctic Notothenioidei, Arctic teleosts have high haemoglobin multiplicity. Although the presence of functionally and structurally distinct haemoglobins is a plesiomorphic condition for many perciform-like fishes, it seems that the oxygen-transport system of teleost fish in the Arctic region has been adjusted to temperature differences and fluctuations of Arctic waters, much larger than in the Antarctic. The amino-acid sequences used to gain insight into the evolution history of α and β globins of polar fish have clearly shown that Antarctic and Arctic globins have different phylogenies, leading to the hypothesis that the selective pressure of environment stability allows the phylogenetic signal to be maintained in the Antarctic sequences, whereas environmental variability would tend to disrupt this signal in Arctic sequences.     

Embryonic β-globin in the non-Antarctic notothenioid fish Cottoperca gobio (Bovichtidae)

Haemoglobins are sensitive to temperature and their properties mirror the thermal conditions encountered by species during their evolutionary histories. This paper provides data on molecular phylogeny of the haemoglobin chains of Cottoperca gobio, a notothenioid fish of sub-Antarctic latitudes, belonging to the basal family Bovichtidae. Unlike most Antarctic notothenioids, C. gobio has two major haemoglobins sharing the β chain. In the molecular phylogenetic analysis, the β chain is included in the clade of the “embryonic” or minor Antarctic globins. Although, in the majority of notothenioids, “embryonic” (minor) α and β globins are expressed in traces or small amounts in the adult stage, in C. gobio the present analysis supports the occurrence of a complete “switch” to exclusive expression of the embryonic β-globin gene in adult fish. The α and β chains sequences have been used to expand our knowledge of the evolution of notothenioid haemoglobins.The protein sequence data reported in this paper will appear in the UniProt Knowledge base under the accession number: P84652 (β chain), P84653 (α ¹ chain).     

Oral rehabilitation of severely worn dentition using an overlay for immediate re-establishment of occlusal vertical dimension

The aim of this study was to describe the treatment used in an elderly patient presenting with bruxism and dental erosion, with good gingival health and bone support, but with decreased occlusal vertical dimension (OVD). The oral rehabilitation of elderly patients presenting with bruxism in association with tooth erosion has been a great challenge for dentists. The loss of OVD, the presence of occlusal instability and the absence of an effective anterior guide due excessive dental wear, can damage stomatognathic system (SS) biology, the function and the aesthetics. In the first treatment stage, an overlay removable partial denture (ORPD) was fabricated for the immediate re-establishment of function and aesthetics. After a 2-month follow up, with the patient presenting no symptoms, a second rehabilitation stage was accomplished, with fixed and removable prostheses. Oral rehabilitation with an ORPD was able to re-establish the SS biology, but a correct diagnosis and treatment plan are essential for success. The ORPD is a non-invasive and reversible restoring modality for general dentists that allow the re-establishment of the patient's immediate aesthetics and function at low cost.     

Are spontaneous fractures possible? An example of clinical application for personalised,multiscale neuro-musculo-skeletal modelling

Elderly frequently present variable degrees of osteopenia, sarcopenia, and neuromotor control degradation. Severely osteoporotic patients sometime fracture their femoral neck when falling. Is it possible that such fractures might occur without any fall, but rather spontaneously while the patient is performing normal movements such as level walking? The aim of this study was to verify if such spontaneous fractures are biomechanically possible, and in such case, which conditions of osteoporosis, sarcopenia, and neuromotor degradation could produce them. To the purpose, a probabilistic multiscale body-organ model validated against controlled experiments was used to predict the risk of spontaneous fractures in a population of 80-years old women, with normal weight and musculoskeletal anatomy, and variable degree of osteopenia, sarcopenia, and neuromotor control degradation. A multi-body inverse dynamics sub-model, coupled to a probabilistic neuromuscular sub-model, and to a femur finite element sub-model, formed the multiscale model, which was run within a Monte Carlo stochastic scheme, where the various parameters were varied randomly according to well defined distributions. The model predicted that neither extreme osteoporosis, nor extreme neuromotor degradation alone are sufficient to predict spontaneous fractures. However, when the two factors are combined an incidence of 0.4% of spontaneous fractures is predicted for the simulated population, which is consistent with clinical reports. When the model represented only severely osteoporotic patients, the incidence of spontaneous fractures increased to 29%. Thus, is biomechanically possible that spontaneous femoral neck fractures occur during level walking, due to a combination of severe osteoporosis and severe neuromotor degradation.     

Three-dimensional reconstruction of tarantula myosin filaments suggests how phosphorylation may regulate myosin activity

Muscle contraction involves the interaction of the myosin heads of the thick filaments with actin subunits of the thin filaments. Relaxation occurs when this interaction is blocked by molecular switches on these filaments. In many muscles, myosin-linked regulation involves phosphorylation of the myosin regulatory light chains (RLCs). Electron microscopy of vertebrate smooth muscle myosin molecules (regulated by phosphorylation) has provided insight into the relaxed structure, revealing that myosin is switched off by intramolecular interactions between its two heads, the free head and the blocked head. Three-dimensional reconstruction of frozen-hydrated specimens revealed that this asymmetric head interaction is also present in native thick filaments of tarantula striated muscle. Our goal in this study was to elucidate the structural features of the tarantula filament involved in phosphorylation-based regulation. A new reconstruction revealed intra- and intermolecular myosin interactions in addition to those seen previously. To help interpret the interactions, we sequenced the tarantula RLC and fitted an atomic model of the myosin head that included the predicted RLC atomic structure and an S2 (subfragment 2) crystal structure to the reconstruction. The fitting suggests one intramolecular interaction, between the cardiomyopathy loop of the free head and its own S2, and two intermolecular interactions, between the cardiac loop of the free head and the essential light chain of the blocked head and between the Leu305-Gln327 interaction loop of the free head and the N-terminal fragment of the RLC of the blocked head. These interactions, added to those previously described, would help switch off the thick filament. Molecular dynamics simulations suggest how phosphorylation could increase the helical content of the RLC N-terminus, weakening these interactions, thus releasing both heads and activating the thick filament.     

Expression of nonstructural rotavirus protein NSP4 mimics Ca2+ homeostasis changes induced by rotavirus infection in cultured cells

Rotavirus infection modifies Ca²⁺ homeostasis, provoking an increase in Ca²⁺ permeation, the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyto), and total Ca²⁺ pools and a decrease in Ca²⁺ response to agonists. A glycosylated viral protein(s), NSP4 and/or VP7, may be responsible for these effects. HT29 or Cos-7 cells were infected by the SA11 clone 28 strain, in which VP7 is not glycosylated, or transiently transfected with plasmids coding for NSP4-enhanced green fluorescent protein (EGFP) or NSP4. The permeability of the plasma membrane to Ca²⁺ and the amount of Ca²⁺ sequestered in the endoplasmic reticulum released by carbachol or ATP were measured in fura-2-loaded cells at the single-cell level under a fluorescence microscope or in cell suspensions in a fluorimeter. Total cell Ca²⁺ pools were evaluated as ⁴⁵Ca²⁺ uptake. Infection with SA11 clone 28 induced an increase in Ca²⁺ permeability and ⁴⁵Ca²⁺ uptake similar to that found with the normally glycosylated SA11 strain. These effects were inhibited by tunicamycin, indicating that inhibition of glycosylation of a viral protein other than VP7 affects the changes of Ca²⁺ homeostasis induced by infection. Expression of NSP4-EGFP or NSP4 in transfected cells induced the same changes observed with rotavirus infection, whereas the expression of EGFP or EGFP-VP4 showed the behavior of uninfected and untransfected cells. Increased ⁴⁵Ca²⁺ uptake was also observed in cells expressing NSP4-EGFP or NSP4, as evidenced in rotavirus infection. These results indicate that glycosylated NSP4 is primarily responsible for altering the Ca²⁺ homeostasis of infected cells through an initial increase of cell membrane permeability to Ca²⁺.     

Galectin-1 in cartilage: Expression, influence on chondrocyte growth and interaction with ECM components

Galectin-1 is a 14 kDa beta-galactoside binding protein, capable of forming lattice-like structures with glycans of cellular glycoconjugates and inducing intracellular signaling. The expression of Galectin-1 in porcine cartilage is described in this work for the first time. Immunocytochemical methods revealed distinct distribution patterns for both articular and growth plate cartilage. In articular cartilage, the highest reactivity for Galectin-1 was found in all chondrocytes at the superficial zone and in most of those at the lower layer of the middle zone. In the growth plate, marked reactivity was seen in chondrocytes at the proliferative zone and reached a maximum level for the column-forming cells at the hypertrophic zone. In addition, different Galectin-1 distribution patterns were observed at the subcellular level. With regards to the metabolic effects of Galectin-1, the results in vitro seem to indicate an inhibitory effect of Galectin-1 on articular chondrocyte anabolism (i.e. inhibition of cell proliferation and anabolic gene expression) and a stimulation of catabolic processes (i.e. induction of matrix degradation and hypertrophy marker expression). These data represent a starting point for the understanding the molecular mechanisms underlining ECM-Galectin-1 interaction and the subsequent signaling-cell transduction processes involving cartilage formation and maturation.