Proteoglycans: key partners in bone cell biology

The diversity of bone proteoglycan (PG) structure and localisation (pericellular, extracellular in the organic bone matrix) reflects a broad spectrum of biological functions within a unique tissue. PGs play important roles in organizing the bone extracellular matrix, taking part in the structuring of the tissue itself as active regulators of collagen fibrillogenesis. PGs also display selective patterns of reactivity with several constituents including cytokines and growth factors, such as transforming growth factor-beta or osteoprotegerin thereby modulating their bio-availability and biological activity in the bone tissue. In this review, the complex PG composition in bone will be addressed together with the specific role played by PGs (or their GAGs chains) in bone biology, as regulatory molecules for bone resorption and their involvement in bone tumor development. These roles have been determined after modulation of PG expression or mutations in their corresponding genes, which revealed specific roles for these compounds in bone pathologies (e.g. perlecan or glypican-3 mutations observed respectively in chondrodysplasia or dysmorphic syndrome). Finally, the potential therapeutic interest of PGs is discussed based on recent data, more particularly on bone tumor-associated osteolysis as these molecules are involved both in bone resorption and tumor development.     

Measurement of Subcellular Force Generation in Neurons

Forces are important for neuronal outgrowth during the initial wiring of the nervous system and after trauma, yet subcellular force generation over the microtubule-rich region at the rear of the growth cone and along the axon has never, to our knowledge, been directly measured. Because previous studies have indicated microtubule polymerization and the microtubule-associated proteins Kinesin-1 and dynein all generate forces that push microtubules forward, a major question is whether the net forces in these regions are contractile or expansive. A challenge in addressing this is that measuring local subcellular force generation is difficult. Here we develop an analytical mathematical model that describes the relationship between unequal subcellular forces arranged in series within the neuron and the net overall tension measured externally. Using force-calibrated towing needles to measure and apply forces, in combination with docked mitochondria to monitor subcellular strain, we then directly measure force generation over the rear of the growth cone and along the axon of chick sensory neurons. We find the rear of the growth cone generates 2.0 nN of contractile force, the axon generates 0.6 nN of contractile force, and that the net overall tension generated by the neuron is 1.3 nN. This work suggests that the forward bulk flow of the cytoskeletal framework that occurs during axonal elongation and growth-cone pauses arises because strong contractile forces in the rear of the growth cone pull material forward.     

Invasive Geotrichum clavatum Fungal Infection in an Acute Myeloid Leukaemia Patient: A Case Report and Review

Invasive Geotrichum clavatum fungal infections are extremely rare and unusual, occurring nearly exclusively in patients experiencing prolonged neutropenia during the treatment for acute myeloid leukaemia. Several groups of cases of fatal G. clavatum infection were reported in France between 2011 and 2012, but the ecological niche has not yet been identified. We report a case of a 32-year-old patient with acute myeloid leukaemia who developed G. clavatum sepsis with primary peritonitis, hepatic nodular lesions, and multivisceral failure during aplasia after induction followed by salvage chemotherapy. He was treated with voriconazole and is still alive 1 year after with controlled disease. We then discuss the epidemiological, clinical, and therapeutic features of these serious fungal infections compared to the published data.     

Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC)

Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.     

Characterization of low density lipoprotein receptor ligand interactions by fluorescence resonance energy transfer

The low density lipoprotein receptor (LDLR) is the prototype of a family of cell surface receptors involved in a wide range of biological processes. A soluble low density lipoprotein receptor (sLDLR) and a tryptophan (Trp)-deficient variant human apolipoprotein E3 (apoE3) N-terminal domain (NT) were used in binding studies. The sole cysteine in apoE3-NT was covalently modified with an extrinsic fluorescence probe, N-(iodoacetyl)-N'-(5-sulfo-1-napthyl)ethylenediamine (AEDANS), and the protein was complexed with lipid. Incubation of sLDLR with AEDANS-Trp-null apoE3-NT dimyristoylphosphatidylcholine (DMPC) disks, but not lipid-free AEDANS-apoE, induced an enhancement in AEDANS fluorescence emission intensity (excitation, 280 nm) consistent with intermolecular energy transfer from excited Trp in sLDLR to receptor-bound apoE. Ligand binding to sLDLR required calcium and was saturable. In competition binding assays, unlabeled apoE3-NT DMPC inhibited AEDANS-apoE DMPC binding to sLDLR more effectively than low density lipoprotein. Fluorescence changes in this system reflected pH-dependent ligand binding and release from sLDLR consistent with models derived from the X-ray crystal structure of the receptor at endosomal pH. Intermolecular energy transfer from excited Trp in LDLR family members to fluorescently tagged ligands represents a sensitive and convenient assay for the characterization of the myriad molecular interactions ascribed to this family of receptor.     

Immunomodulatory effects of probiotics in the intestinal tract

The intestinal microbiota is the largest source of microbial stimulation that exerts both harmful and beneficial effects on human health. The interaction between probiotic and enterocytes is the initiating event in immunomodulation and merits particular attention. The effects of probiotic is strain dependent and for each new probiotic strain, profiles of cytokines secreted by lymphocytes, enterocytes or dendritic cells that come in contact with the strain should be systematically established. To evaluate the effects of probiotics on the immune system, models that mimic the mucosa, and thus the physiological reality, should be preferred whenever it is possible. Then, the in vitro observed effects should be backed up by properly conducted randomized double bind clinical studies. More detailed studies are needed to determine the precise action mode of probiotics on both mucosal and systemic immunity.