Neuropeptide Y Attenuates Stress‐Induced Bone Loss Through Suppression of Noradrenaline Circuits

Chronic stress and depression have adverse consequences on many organ systems, including the skeleton, but the mechanisms underlying stress‐induced bone loss remain unclear. Here we demonstrate that neuropeptide Y (NPY), centrally and peripherally, plays a critical role in protecting against stress‐induced bone loss. Mice lacking the anxiolytic factor NPY exhibit more anxious behavior and elevated corticosterone levels. Additionally, following a 6‐week restraint, or cold‐stress protocol, Npy‐null mice exhibit three‐fold greater bone loss compared to wild‐type mice, owing to suppression of osteoblast activity. This stress‐protective NPY pathway acts specifically through Y2 receptors. Centrally, Y2 receptors suppress corticotropin‐releasing factor expression and inhibit activation of noradrenergic neurons in the paraventricular nucleus. In the periphery, they act to control noradrenaline release from sympathetic neurons. Specific deletion of arcuate Y2 receptors recapitulates the Npy‐null stress response, coincident with elevated serum noradrenaline. Importantly, specific reintroduction of NPY solely in noradrenergic neurons of otherwise Npy‐null mice blocks the increase in circulating noradrenaline and the stress‐induced bone loss. Thus, NPY protects against excessive stress‐induced bone loss, through Y2 receptor‐mediated modulation of central and peripheral noradrenergic neurons. © 2014 American Society for Bone and Mineral Research.     

Adverse effects of perioperative paracetamol,NSAIDs, glucocorticoids,gabapentinoids and their combinations: a topical review

Post‐operative pain affects millions of patients worldwide and the post‐operative period has high rates of morbidity and mortality. Some of this morbidity may be related to analgesics. The aim of this review was to provide an update of current knowledge of adverse events (AE) associated with the most common perioperative non‐opioid analgesics: paracetamol, non‐steroidal anti‐inflammatory drugs (NSAIDs), glucocorticoids (GCCs), gabapentinoids and their combinations. The review is based on data from systematic reviews with meta‐analyses of analgesic efficacy and/or adverse effects of perioperative non‐opioid analgesics, and randomised trials and cohort/retrospective studies. Generally, data on AE are sparse and related to the immediate post‐operative period. For paracetamol, the incidence of AEs appears trivial. Data are inconclusive regarding an association of NSAIDs with mortality, cardiovascular events, surgical bleeding and renal impairment. Anastomotic leakage may be associated with NSAID usage. No firm evidence exists for an association of NSAIDs with impaired bone healing. Single‐dose GCCs were not significantly related to increased infection rates or delayed wound healing. Gabapentinoid treatment was associated with increased sedation, dizziness and visual disturbances, but the clinical relevance needs clarification. Importantly, data on AEs of combinations of the above analgesics are sparse and inconclusive. Despite the potential adverse events associated with the most commonly applied non‐opioid analgesics, including their combinations, reporting of such events is sparse and confined to the immediate perioperative period. Knowledge of benefit and harm related to multimodal pain treatment is deficient and needs clarification in large trials with prolonged observation.     

The use of 7-amino actinomycin D in identifying apoptosis: simplicity of use and broad spectrum of application compared with other techniques

The detection and quantitation of apoptotic cells is becoming increasingly important in the investigation of the role of apoptosis in cellular proliferation and differentiation. The pathogenesis of hematologic disorders such as aplastic anemia and the development of neoplasia are believed to involve dysregulation of apoptosis. To quantitate accurately the proportion of apoptosis cells within different cell types of a heterogeneous cell population such as blood or bone marrow, a method is required that combines the analysis of large numbers of cells with concurrent immunophenotyping of cell surface antigens. In this study, we have evaluated such a method using the fluorescent DNA binding agent, 7-amino actinomycin D (7AAD), to stain three diverse human cell lines, induced to undergo apoptosis by three different stimuli. Flow cytometric analysis defines three populations on the basis of 7AAD fluorescence and forward light scatter. We have shown by cell sorting and subsequent morphological assessment and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling that the populations defined by 7AAD represent live, apoptotic, and late-apoptotic/dead cells. This method is quick, simple, reproducible, and cheap and will be a valuable tool in the investigation of the role of apoptosis in normal physiology and in disease states.     

Three residues at the interface of factor XI (FXI) monomers augment covalent dimerization of FXI

Summary.  Background:  Human plasma factor XI is a homodimer, with each monomer comprising a catalytic domain and four homologous 'apple' domains. The monomers bind to each other through non-covalent bonds and through a disulfide bond between Cys321 residues in apple 4 domains. Objective:  To identify residues essential for dimerization in the FXI monomer interface. Methods:  Specificity-determining residues in apple 4 domains were sought by sequence alignment of FXI and prekallikrein apple domains in different species. Specific residues identified in apple 4 domains were mutagenized and expressed in baby hamster kidney (BHK) cells for evaluation of their effect on FXI dimerization, analyzed by non-reduced sodium dodecylsulfate polyacrylamide gel electrophoresis and size-exclusion chromatography. Results:  Among the 19 residues of the FXI monomer interface, Leu284, Ile290 and Tyr329 were defined as specificity-determining residues. Substitutions of these residues or pairs of residues did not affect FXI synthesis and secretion from transfected BHK cells, but did impair dimerization, despite the presence of cysteine at position 321. The double mutant 284A/290A yielded predominantly a monomer, whereas all other single or double mutants yielded monomers as well as disulfide-bonded dimers. Conclusions:  The data suggest that Leu284, Ile290 and Tyr329 in the interface of FXI monomers are essential for forming non-covalently bonded dimers that facilitate formation of a disulfide-bonded stable FXI dimer.