CGMD Platform: Integrated Web Servers for the Preparation, Running, and Analysis of Coarse-Grained Molecular Dynamics Simulations.

Advances in coarse-grained molecular dynamics (CGMD) simulations have extended the use of computational studies on biological macromolecules and their complexes, as well as the interactions of membrane protein and lipid complexes at a reduced level of representation, allowing longer and larger molecular dynamics simulations. Here, we present a computational platform dedicated to the preparation, running, and analysis of CGMD simulations. The platform is built on a completely revisited version of our Martini coarsE gRained MembrAne proteIn Dynamics (MERMAID) web server, and it integrates this with other three dedicated services. In its current version, the platform expands the existing implementation of the Martini force field for membrane proteins to also allow the simulation of soluble proteins using the Martini and the SIRAH force fields. Moreover, it offers an automated protocol for carrying out the backmapping of the coarse-grained description of the system into an atomistic one.

DEPDC1/LET-99 participates in an evolutionarily conserved pathway for anti-tubulin drug-induced apoptosis.

Microtubule-targeting chemotherapeutics induce apoptosis in cancer cells by promoting the phosphorylation and degradation of the anti-apoptotic BCL-2 family member MCL1. The signalling cascade linking microtubule disruption to MCL1 degradation remains however to be defined. Here, we establish an in vivo screening strategy in Caenorhabditis elegans to uncover genes involved in chemotherapy-induced apoptosis. Using an RNAi-based screen, we identify three genes required for vincristine-induced apoptosis. We show that the DEP domain protein LET-99 acts upstream of the heterotrimeric G protein alpha subunit GPA-11 to control activation of the stress kinase JNK-1. The human homologue of LET-99, DEPDC1, similarly regulates vincristine-induced cell death by promoting JNK-dependent degradation of the BCL-2 family protein MCL1. Collectively, these data uncover an evolutionarily conserved mediator of anti-tubulin drug-induced apoptosis and suggest that DEPDC1 levels could be an additional determinant for therapy response upstream of MCL1.

Culturing muscle fibres in hanging drop: a novel approach to solve an old problem.

BACKGROUND INFORMATION: The satellite cells (SCs) associated with muscle fibres play a key role in postnatal growth and regeneration of skeletal muscle. Commonly used methods of isolation and in vitro culture of SCs lead to the mixture of their subpopulations that exist within muscle. To solve this problem, we used the well established technique, the hanging drop system, to culture SCs in a three-dimensional environment and thus, to monitor them in their original niche. RESULTS: Using hanging drop technique, we were able to culture SCs associated with the fibre at least for 9 days with one transfer of fibres to the fresh drops. In comparison, in the classical method of myofibres culture, that is, on the dishes coated with Matrigel, SCs leave the fibres within 3 days after the isolation. Cells cultured in both systems differed in expression of Pax7 and MyoD. While almost all cells cultured in adhesion system expressed MyoD before the fifth day of the culture, the majority of SCs cultured in hanging drop still maintained expression of Pax7 and were not characterised by the presence of MyoD. Among the cells cultured with single myofibre for up to 9 days, we identified two different subclones of SCs: low proliferative clone and high proliferative clone, which differed in proliferation rate and membrane potential. CONCLUSIONS: The hanging drop enables the myofibres to be kept in suspension for at least 9 days, and thus, allows SCs and their niche to interact each other for prolonged time. In a consequence, SCs cultured in hanging drop maintain expression of Pax7 while those cultured in a traditional adhesion culture, that is, devoid of signals from the original niche, activate and preferentially undergo differentiation as manifested by expression of MyoD. Thus, the innovative method of SCs culturing in the hanging drop system may serve as a useful tool to study the fate of different subpopulations of these cells in their anatomical location and to determine reciprocal interactions between them and their niche.

Loss of CB1 receptors leads to decreased cathepsin D levels and accelerated lipofuscin accumulation in the hippocampus.

Early onset of age-related changes in the brain of cannabinoid 1 receptor knockout (Cnr1(-/-)) mice suggests that cannabinoid 1 (CB1) receptor activity significantly influences the progression of brain aging. In the present study we show that lack of CB1 receptors leads to a significant increase in lipofuscin accumulation and a reduced expression and activity of cathepsin D, lysosomal protease implicated in the degradation of damaged macromolecules, in the hippocampus of 12-month-old mice. The impaired clearance of damaged macromolecules due to the low cathepsin D levels and not enhanced oxidative stress may be responsible for the lipofuscin accumulation because macromolecule oxidation levels were comparable between the genotypes within the same age group. The altered levels of autophagy markers p62 and LC3-II suggest that autophagy is upregulated in CB1 knockout mice. Increased autophagic flux in the absence of CB1 receptors is probably a compensatory mechanism to partially counteract decreased lysosomal degradation capacity. Together, these results suggest that CB1 receptor activity affects lysosomal activity, degradation of damaged macromolecules and thus it may influence the course and onset of brain aging.

In vivo tissue engineering of functional skeletal muscle by freshly isolated satellite cells embedded in a photopolymerizable hydrogel.

The success of skeletal muscle reconstruction depends on finding the most effective, clinically suitable strategy to engineer myogenic cells and biocompatible scaffolds. Satellite cells (SCs), freshly isolated or transplanted within their niche, are presently considered the best source for muscle regeneration. Here, we designed and developed the delivery of either SCs or muscle progenitor cells (MPCs) via an in situ photo-cross-linkable hyaluronan-based hydrogel, hyaluronic acid-photoinitiator (HA-PI) complex. Partially ablated tibialis anterior (TA) of C57BL/6J mice engrafted with freshly isolated satellite cells embedded in hydrogel showed a major improvement in muscle structure and number of new myofibers, compared to muscles receiving hydrogel + MPCs or hydrogel alone. Notably, SCs embedded in HA-PI also promoted functional recovery, as assessed by contractile force measurements. Tissue reconstruction was associated with the formation of both neural and vascular networks and the reconstitution of a functional SC niche. This innovative approach could overcome previous limitations in skeletal muscle tissue engineering.

Three-dimensional porous scaffold allows long-term wild-type cell delivery in dystrophic muscle.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin; affected muscles are characterized by continuous bouts of muscle degeneration, eventually leading to the exhaustion of the endogenous satellite cell pool. At present, only palliative treatments are available, although several gene and cell therapy-based approaches are being studied. In this study we proposed to overcome the limitations hampering intramuscular cell injection by using a biomaterial-based strategy. In particular, we used a three-dimensional (3D) collagen porous scaffold to deliver myogenic precursor cells (MPCs) in vivo in the mdx murine model of DMD. MPCs, derived from single fibres of wild-type donors, were expanded in vitro, seeded onto collagen scaffolds and implanted into the tibialis anterior muscles of normal and mdx mice. As a control, cells were delivered via direct intramuscular cell injection in the contralateral muscles. Scaffold-delivered MPCs displayed lower apoptosis and higher proliferation than injected cells; in terms of dystrophin restoration, collagen scaffolds yielded better results than direct injections. Importantly, time-course experiments indicated that the scaffolds acted as a cell reservoir, although cell migration was mostly contained within 400 µm from the scaffold-host tissue interface.

Advances in musculoskeletal tissue engineering: moving towards therapy.

Skeletal muscle can self-repair, but is unable to restore significant tissue loss, as consequence of trauma, congenital defects, tumor ablation or denervation. Intramuscular injection of autologous or allogenic derived myogenic cells (namely satellite cells and myoblasts) did not lead to efficient regeneration because of poor cell retention and survival, as well as immunorejection. In the last decade, tissue engineering looked at overcoming these problems by investigating alternative treatment options, i.e., the suspension of myogenic precursors in temporary matrix, formed by biodegradable and biocompatible materials. This approach allows to engineer custom architectured preconditioned implants, and locally deliver paracrine factors.This article reviews current and potential strategies for the repair of damaged muscle and suggests some innovative approaches for the translation to the clinical setting.

Amniotic fluid stem cell migration after intraperitoneal injection in pup rats: implication for therapy.

PURPOSE: Despite being commonly used in clinical practice, the intraperitoneal (i.p.) route has been rarely used for cell delivery. We evaluated the capacity of amniotic fluid stem (AFS) cells, administered i.p., to diffuse systemically and to integrate into tissues of healthy newborn rats. METHODS: AFS cells were obtained from pregnant GFP + Sprague-Dawley rats by c-kit selection. Wild-type Sprague-Dawley newborn rats were divided into two groups receiving i.p.: (1) 2 x 10(6) AFS cells (n = 12); (2) of phosphate buffer saline (PBS) (n = 2) at 24 and 48 h after birth. Animals were either killed at 96 h of life, and organs collected for gfp amplification, or at 3 weeks of life and tissues isolated for green fluorescence protein (GFP) immunofluorescence. RESULTS: No adverse effects were observed after i.p. injection of PBS or AFS cells. Gfp was amplified in at least one organ in all rats injected with AFS cells except one (11/12). The intestine was the organ found most frequently positive (67%) followed by liver (25%), spleen (16%), heart (16%), lungs (16%), femur (8%) and brain (0%). Immunohistochemistry confirmed PCR results. CONCLUSION: In the short term, the i.p. administration of AFS cells, is a safe procedure and allows their migration, homing and integration into various organs of healthy newborn rats.

Cobalt induces oxidative stress in isolated liver mitochondria responsible for permeability transition and intrinsic apoptosis in hepatocyte primary cultures.

It is well established that cobalt mediates the occurrence of oxidative stress which contributes to cell toxicity and death. However, the mechanisms of these effects are not fully understood. This investigation aimed at establishing if cobalt acts as an inducer of mitochondrial-mediated apoptosis and at clarifying the mechanism of this process. Cobalt, in the ionized species Co(2+), is able to induce the phenomenon of mitochondrial permeability transition (MPT) in rat liver mitochondria (RLM) with the opening of the transition pore. In fact, Co(2+) induces mitochondrial swelling, which is prevented by cyclosporin A and other typical MPT inhibitors such as Ca(2+) transport inhibitors and bongkrekic acid, as well as anti-oxidant agents. In parallel with mitochondrial swelling, Co(2+) also induces the collapse of electrical membrane potential. However in this case, cyclosporine A and the other MPT inhibitors (except ruthenium red and EGTA) only partially prevent DeltaPsi drop, suggesting that Co(2+) also has a proton leakage effect on the inner mitochondrial membrane. MPT induction is due to oxidative stress, as a result of generation by Co(2+) of the highly damaging hydroxyl radical, with the oxidation of sulfhydryl groups, glutathione and pyridine nucleotides. Co(2+) also induces the release of the pro-apoptotic factors, cytochrome c and AIF. Incubation of rat hepatocyte primary cultures with Co(2+) results in apoptosis induction with caspase activation and increased level of expression of HIF-1alpha. All these observations allow us to state that, in the presence of calcium, Co(2+) is an inducer of apoptosis triggered by mitochondrial oxidative stress.

Different cardiovascular potential of adult- and fetal-type mesenchymal stem cells in a rat model of heart cryoinjury.

Efficacy of adult (bone marrow, BM) versus fetal (amniotic fluid, AF) mesenchymal stem cells (MSCs) to replenish damaged rat heart tissues with new cardiovascular cells has not yet been established. We investigated on the differentiation potential of these two rat MSC populations in vitro and in a model of acute necrotizing injury (ANI) induced by cryoinjury. Isolated BM-MSCs and AF-MSCs were characterized by flow cytometry and cytocentrifugation and their potential for osteogenic, adipogenic, and cardiovascular differentiation assayed in vitro using specific induction media. The left anterior ventricular wall of syngeneic Fisher 344 (n = 48) and athymic nude (rNu) rats (n = 6) was subjected to a limited, nontransmural epicardial ANI in the approximately one third of wall thickness without significant hemodynamic effects. The time window for in situ stem cell transplantation was established at day 7 postinjury. Fluorochrome (CMTMR)-labeled BM-MSCs (2 x 10(6)) or AF-MSCs (2 x 10(6)) were injected in syngeneic animals (n = 26) around the myocardial lesion via echocardiographic guidance. Reliability of CMTMR cell tracking in this context was ascertained by transplanting genetically labeled BM-MSCs or AF-MSCs, expressing the green fluorescent protein (GFP), in rNu rats with ANI. Comparison between the two methods of cell tracking 30 days after cell transplantation gave slightly different values (1420,58 +/- 129,65 cells/mm2 for CMTMR labeling and 1613.18 +/- 643.84 cells/mm2 for genetic labeling; p = NS). One day after transplantation about one half CMTMR-labeled AF-MSCs engrafted to the injured heart (778.61 +/- 156.28 cells/mm2) in comparison with BM-MSCs (1434.50 +/- 173.80 cells/mm2, p < 0.01). Conversely, 30 days after cell transplantation survived MSCs were similar: 1275.26 +/- 74.51/mm2 (AF-MSCs) versus 1420.58 +/- 129.65/mm2 for BM-MSCs (p = NS). Apparent survival gain of AF-MSCs between the two time periods was motivated by the cell proliferation rate calculated at day 30, which was lower for BM-MSCs (6.79 +/- 0.48) than AF-MSCs (10.83 +/- 3.50; p < 0.01), in the face of a similar apoptotic index (4.68 +/- 0.20 for BM-MSCs and 4.16 +/- 0.58 for AF-MSCs; p = NS). These cells were also studied for their expression of markers specific for endothelial cells (ECs), smooth muscle cells (SMCs), and cardiomyocytes (CMs) using von Willebrand factor (vWf), smooth muscle (SM) alpha-actin, and cardiac troponin T, respectively. Grafted BM-MSCs or AF-MSCs were found as single cell/small cell clusters or incorporated in the wall of microvessels. A larger number of ECs (227.27 +/- 18.91 vs. 150.36 +/- 24.08 cells/mm2, p < 0.01) and CMs (417.91 +/- 100.95 vs. 237.43 +/- 79.99 cells/mm2, p < 0.01) originated from AF-MSCs than from BM-MSCs. Almost no SMCs were seen with AF-MSCs, in comparison to BM-MSCs (98.03 +/- 40.84 cells/mm2), in concordance with lacking of arterioles, which, instead, were well expressed with BM-MSCs (71.30 +/- 55.66 blood vessels/mm2). The number of structurally organized capillaries was slightly different with the two MSCs (122.49 +/- 17.37/mm2 for AF-MSCs vs. 148.69 +/- 54.41/mm2 for BM-MSCs; p = NS). Collectively, these results suggest that, in the presence of the same postinjury microenvironment, the two MSC populations from different sources are able to activate distinct differentiation programs that potentially can bring about a myocardial-capillary or myocardial-capillary-arteriole reconstitution.

Glycyrrhetinic acid as inhibitor or amplifier of permeability transition in rat heart mitochondria.

Glycyrrhetinic acid (GE), a hydrolysis product of glycyrrhizic acid, one of the main constituents of licorice root, is able, depending on its concentration, to prevent or to induce the mitochondrial permeability transition (MPT) (a phenomenon related to oxidative stress) in rat heart mitochondria (RHM). In RHM, below a threshold concentration of 7.5 microM, GE prevents oxidative stress and MPT induced by supraphysiological Ca2+ concentrations. Above this concentration, GE induces oxidative stress by interacting with a Fe-S centre of Complex I, thus producing ROS, and amplifies the opening of the transition pore, once again induced by Ca2+. GE also inhibits Ca2+ transport in RHM, thereby preventing the oxidative stress induced by the cation. However, the reduced amount of Ca2+ transported in the matrix is sufficient to predispose adenine nucleotide translocase for pore opening. Comparisons between observed results and the effects of GE in rat liver mitochondria (RLM), in which the drug induces only MPT without exhibiting any protective effect, confirm that it interacts in a different way with RHM, suggesting tissue specificity for its action. The concentration dependence of the opposite effects of GE, in RHM but not RLM, is most probably due to the existence of a different, more complex, pathway by means of which GE reaches its target. It follows that high GE concentrations are necessary to stimulate the oxidative stress capable of inducing MPT, because of the above effect, which prevents the interaction of low concentrations of GE with the Fe-S centre. The reported results also explain the mechanism of apoptosis induction by GE in cardiomyocytes.