Looking for Calcium Phosphate Composite Suitable to Study Osteoclast Endocytosis: Preliminary Observations.

One of the issues regarding in vitro study of bone resorption is the synthesis of a bone-like biomaterial forming a thin layer onto either glass or plastic. The synthesis of a bone-like material suitable for in vitro studies can be valuable both to investigate osteoclast differentiation, that in vivo proceeds within the local microenvironment of bone and to understand how its presence triggers activation of macrophages present in situ when bone is damaged (a scenario that can occur for example in case of bone fracture). Despite the intensive studies committed to recreate synthetic bone analogues, the most used substrates for in vitro studies on bone resorption are slices of bone or dentine. Therefore morphological investigations (i.e. fluorescence analysis and phase contrast) are strongly compromised due to the thickness of the bone analogue. In the present study, with the aim to guarantee a versatile (and easy to be made) substrate, that could be suitable to study cell adhesion and morphology by epifluorescence, phase contrast and TEM, we developed a biomaterial containing a calcium phosphate salt and type I collagen. This material (made specifically for in vitro studies) forms a very thin layer that allowed to merge the morphological information derived from phase-contrast and epifluorescence observation, making possible the observation of the interface between cell and matrix. Moreover the electron microscopy evaluation of the endocytosis performed on cell differentiated could be more suitable because sample does not need the process of demineralization.

Towards a full retrieval of the deformation tensor F using convergent beam electron diffraction.

A new method to retrieve the local lattice parameters and rotations in a crystal from off-axis convergent beam electron diffraction (CBED) patterns is presented and validated using Bloch wave dynamical simulations. The originality of the method is to use both the diffracted and transmitted beams and to use kinematical approximations in the fitting algorithm. The study is based on the deformation gradient tensor F which includes rotation and strain. Working on simulated images it is shown that (i) from a single direction of observation, seven parameters out of the nine parameters of F can be determined with an accuracy of 3 × 10(-4) for the normal strain parameters εxx, εyy, and εzz, (ii) the unit cell volume can only be retrieved if the diffracted and transmitted beams are both included in the fitting and (iii) all the nine parameters of F can be determined by combining two directions of observation separated by about 20°.

NH2-truncated human tau induces deregulated mitophagy in neurons by aberrant recruitment of Parkin and UCHL-1: implications in Alzheimer's disease.

Disarrangement in functions and quality control of mitochondria at synapses are early events in Alzheimer's disease (AD) pathobiology. We reported that a 20-22 kDa NH2-tau fragment mapping between 26 and 230 amino acids of the longest human tau isoform (aka NH2htau): (i) is detectable in cellular and animal AD models, as well in synaptic mitochondria and cerebrospinal fluids (CSF) from human AD subjects; (ii) is neurotoxic in primary hippocampal neurons; (iii) compromises the mitochondrial biology both directly, by inhibiting the ANT-1-dependent ADP/ATP exchange, and indirectly, by impairing their selective autophagic clearance (mitophagy). Here, we show that the extensive Parkin-dependent turnover of mitochondria occurring in NH2htau-expressing post-mitotic neurons plays a pro-death role and that UCHL-1, the cytosolic Ubiquitin-C-terminal hydrolase L1 which directs the physiological remodeling of synapses by controlling ubiquitin homeostasis, critically contributes to mitochondrial and synaptic failure in this in vitro AD model. Pharmacological or genetic suppression of improper mitophagy, either by inhibition of mitochondrial targeting to autophagosomes or by shRNA-mediated silencing of Parkin or UCHL-1 gene expression, restores synaptic and mitochondrial content providing partial but significant protection against the NH2htau-induced neuronal death. Moreover, in mitochondria from human AD synapses, the endogenous NH2htau is stably associated with Parkin and with UCHL-1. Taken together, our studies show a causative link between the excessive mitochondrial turnover and the NH2htau-induced in vitro neuronal death, suggesting that pathogenetic tau truncation may contribute to synaptic deterioration in AD by aberrant recruitment of Parkin and UCHL-1 to mitochondria making them more prone to detrimental autophagic clearance.

AD-linked, toxic NH2 human tau affects the quality control of mitochondria in neurons.

Functional as well as structural alterations in mitochondria size, shape and distribution are precipitating, early events in progression of Alzheimer's Disease (AD). We reported that a 20-22kDa NH2-tau fragment (aka NH2htau), mapping between 26 and 230 amino acids of the longest human tau isoform, is detected in cellular and animal AD models and is neurotoxic in hippocampal neurons. The NH2htau -but not the physiological full-length protein- interacts with Aß at human AD synapses and cooperates with it in inhibiting the mitochondrial ANT-1-dependent ADP/ATP exchange. Here we show that the NH2htau also adversely affects the interplay between the mitochondria dynamics and their selective autophagic clearance. Fragmentation and perinuclear mislocalization of mitochondria with smaller size and density are early found in dying NH2htau-expressing neurons. The specific effect of NH2htau on quality control of mitochondria is accompanied by (i) net reduction in their mass in correlation with a general Parkin-mediated remodeling of membrane proteome; (ii) their extensive association with LC3 and LAMP1 autophagic markers; (iii) bioenergetic deficits and (iv) in vitro synaptic pathology. These results suggest that NH2htau can compromise the mitochondrial biology thereby contributing to AD synaptic deficits not only by ANT-1 inactivation but also, indirectly, by impairing the quality control mechanism of these organelles.

Tanshinone VI inhibits the expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1.

This study investigated the possible antitumor mechanisms of action of Tanshinone VI, one of the components of Salvia miltiorrhiza Bunge, which is used in traditional Chinese herbal medicine. To this end, the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), were evaluated in-vitroin tumor necrosis factor-alpha (TNF-alpha)-stimulated endothelial cells, with, or without the addition of Tanshinone VI (10, 20, 30, or 40 mM) in the culture medium; the effects of Tanshinone VI on angiogenesis was also evaluated with an epithelial cell tube formation assay and its toxicity was evaluated with a colorimetric (MTT) cell viability assay. The results showed that the up-regulation of ICAM-1 and VCAM-1 induced by TNF-alpha was dose-dependently inhibited by Tanshinone VI, with restoration of control levels at the dose of 40 mM; Tanshinone VI also had a remarkable anti-angiogenesis effect, already at the dose of 10 mM, while none of the doses tested had significant effects on cell viability. These results indicate that the antitumor properties of Tanshinone VI can be ascribed to the inhibition of cell adhesion, due to blockage of the up-regulation of cell adhesion molecules, with the consequent inhibition of metastases formation and/or angiogenesis. The lack of toxic effects at the dosage used makes Tanshinone VI a good candidate for its therapeutic use in humans.

Time-dependent relaxation of strained silicon-on-insulator lines using a partially coherent x-ray nanobeam.

We report on the quantitative determination of the strain map in a strained silicon-on-insulator line with a 200×70 nm2 cross section. In order to study a single line as a function of time, we used an x-ray nanobeam with relaxed coherence properties as a compromise between beam size, coherence, and intensity. We demonstrate how it is possible to refine the line deformation map at the nanoscale, and follow its evolution as the line relaxes under the influence of the x-ray nanobeam. We find that the strained line flattens itself under irradiation but maintains the same linear strain (ε(zz) unchanged).

Production of in vivo-biotinylated rotavirus particles.

Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6-BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6-BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

BAG3 controls angiogenesis through regulation of ERK phosphorylation.

BAG3 is a co-chaperone of the heat shock protein (Hsp) 70, is expressed in many cell types upon cell stress, however, its expression is constitutive in many tumours. We and others have previously shown that in neoplastic cells BAG3 exerts an anti-apoptotic function thus favoring tumour progression. As a consequence we have proposed BAG3 as a target of antineoplastic therapies. Here we identify a novel role for BAG3 in regulation of neo-angiogenesis and show that its downregulation results in reduced angiogenesis therefore expanding the role of BAG3 as a therapeutical target. In brief we show that BAG3 is expressed in endothelial cells and is essential for the interaction between ERK and its phosphatase DUSP6, as a consequence its removal results in reduced binding of DUSP6 to ERK and sustained ERK phosphorylation that in turn determines increased levels of p21 and p15 and cell-cycle arrest in the G1 phase.

Coherent x-ray wavefront reconstruction of a partially illuminated Fresnel zone plate.

A detailed characterization of the coherent x-ray wavefront produced by a partially illuminated Fresnel zone plate is presented. We show, by numerical and experimental approaches, how the beam size and the focal depth are strongly influenced by the illumination conditions, while the phase of the focal spot remains constant. These results confirm that the partial illumination can be used for coherent diffraction experiments. Finally, we demonstrate the possibility of reconstructing the complex-valued illumination function by simple measurement of the far field intensity in the specific case of partial illumination.

Inhibition of bone resorption by Tanshinone VI isolated from Salvia miltiorrhiza Bunge.

During the last decade, a more detailed knowledge of molecular mechanisms involved in osteoclastogenesis has driven research efforts in the development and screening of compound libraries of several small molecules that specifically inhibit the pathway involved in the commitment of the osteoclast precursor cells. Natural compounds that suppress osteoclast differentiation may have therapeutic value in treating osteoporosis and other bone erosive diseases such as rheumatoid arthritis or metastasis associated with bone loss. In ongoing investigation into anti-osteoporotic compounds from natural products we have analyzed the effect of Tanshinone VI on osteoclasts differentiation, using a physiologic three-dimensional osteoblast/bone marrow model of cell co-culture. Tanshinone VI is an abietane diterpene extracted from the root of Salvia miltiorrhiza Bunge (Labiatae), a Chinese traditional crude drug, "Tan-Shen". Tashinone has been widely used in clinical practice for the prevention of cardiac diseases, arthritis and other inflammation-related disorders based on its pharmacological actions in multiple tissues. Although Tanshinone VI A has been used as a medicinal agent in the treatment of many diseases, its role in osteoclast-related bone diseases remains unknown. We showed previously that Tanshinone VI greatly inhibits osteoclast differentiation and suppresses bone resorption through disruption of the actin ring; subsequently, we intended to examine the precise inhibitory mechanism of Tanshinone VI on osteoclast differentiating factor. This study shows, for the first time, that Tanshinone VI prevents osteoclast differentiation by inhibiting RANKL expression and NFkB induction.

Clathrin-dependent endocytosis of membrane-bound RANKL in differentiated osteoclasts.

Bone is continuously repaired and remodelled through well-coordinated activity of osteoblasts that form new bone and osteoclasts, which resorb it. Osteoblasts synthesize and secrete two key molecules that are important for osteoclast differentiation, namely the ligand for the receptor of activator of nuclear factor kappaB (RANKL) and its decoy receptor osteoprotegerin (OPG). Active membrane transport is a typical feature of the resorbing osteoclast during bone resorption. Normally, one resorption cycle takes several hours as observed by monitoring actin ring formation and consequent disappearance in vitro. During these cyclic changes, the cytoskeleton undergoes remarkable dynamic rearrangement. Active cells show a continuous process of exocytosis that plays an essential role in transport of membrane components, soluble molecules and receptor-mediated ligands thus allowing them to communicate with the environment. The processes that govern intracellular transport and trafficking in mature osteoclasts are poorly known. The principal methodological problem that have made these studies difficult is a physiological culture of osteoclasts that permit observing the vesicle apparatus in conditions similar to the in vivo conditions. In the present study we have used a number of morphological approaches to characterize the composition, formation and the endocytic and biosynthetic pathways that play roles in dynamics of differentiation of mature bone resorbing cells using a tri-dimensional system of physiologic coculture.

Effect of recombinant TRAIL in a murine co-culture system of osteoclastogenesis.

Although some experimental evidence has implicated the TRAIL/TRAIL-receptor system in the regulation of osteoclastogenesis, the only available studies performed so far have been performed on isolated pre-osteoclasts, induced to differentiate by the addition of recombinant RANKL and M-CSF. Using a more physiological co-culture system in the absence of exogenous cytokines, we have here demonstrated that recombinant TRAIL inhibits osteoclast formation, but only at relatively high concentrations (500 ng/mL).

Strain, size, and composition of InAs quantum sticks embedded in InP determined via grazing incidence x-ray anomalous diffraction.

We have used x-ray anomalous diffraction to recover the model-independent Fourier transform (x-ray structure factor) of InAs quantum sticklike islands embedded in InP. The average height of the quantum sticks, as deduced from the width of the structure factor profile, is 2.54 nm. The InAs out-of-plane deformation, relative to InP, is 6.1%. Diffraction anomalous fine structure provides evidence of pure InAs quantum sticks. Finite difference method calculations reproduce well the diffraction data, and give the strain along the growth direction. The chemical mixing at interfaces is also analyzed.

The adhesion of Flow 2002 fibroblasts to titanium implant materials is influenced by different surface topographies and is related to the immunocytochemical expression of fibronectin.

Osteointegrated titanium dental implants are widely used biomaterials that have to integrate within the alveolar bone and interact with periodontal soft tissues. In this study, we investigated the immunocytochemical expression of the extra-cellular matrix (ECM) protein fibronectin (FN) and type I collagen (Coll I) in Flow 2002 fibroblast cultures spread on grade III-titanium samples with five different surface topographies and we correlated the immunocytochemical data to the adhesion capability of these cells to the above-mentioned substrates. Five different surfaces of grade III-titanium implants were at first characterized both by scanning electron microscopy (SEM) and by laser profilometry for surface roughness evaluation. After being spread on the biomaterial surfaces, the fibroblasts were left to proliferate for 72 hr and subsequently the cells underwent immunocytochemical procedures for detecting both FN and Coll I. The fibroblasts appeared more adherent to smoother titanium surfaces than to rougher ones; however, the highest cell density was detected on the roughest surface, even if it was unrelated to the highest FN expression. In the other biomaterial surfaces examined, as well as in controls, immunocytochemical FN expression correlated effectively to cell density on the examined substratum, whereas no determinant information was available regarding Coll I. It is reasonable to assume that surface roughness could be a relevant parameter influencing fibroblast adhesion to substrata; however, the evaluation of the cell density only is insufficient, and should be supported by the immunocytochemical FN expression, which could be confirmed as a useful tool in determining implant material biocompatibility. (Journal of Applied Biomaterials & Biomechanics 2004; 2: 169-76).

Structural evidence for Ta-tetramerization displacements in the charge-density-wave compound (TaSe4)2I from x-ray anomalous diffraction.

We use the anomalous x-ray diffraction technique to investigate the nature of the tantalum displacement pattern in the modulated phase of the charge-density-wave compound (TaSe4)2I. In addition to the known acousticlike modulation, we find the first direct evidence for the condensation of opticlike Ta displacements along the metallic chains corresponding to an LLSS pattern of long and short in-chain Ta-Ta distances (Ta-tetramerization modes). This result confirms a previous model in which the interaction of the electronically coupled optic modes with long-wavelength acoustic shear modes leads to the condensation of a modulation of mixed character.