Paolo Di Nardo

Engineering materials and biology to boost performance of microbial fuel cells: a critical review

In less than a decade the levels of performance of microbial fuel cells (MFCs) in terms of current output, voltage, and power density have grown tremendously according to steady exponential trends. Achievements occurred over the past 2–3 years have been particularly impressive. This is due partly to a better understanding of the biological aspects of this multidisciplinary technology, but also to systematic work undertaken by several research groups worldwide aimed at improving and optimizing aspects related to materials and system configuration. Aim of this review is to outline the current perspective about MFCs by focusing on the recent major advances in the areas of materials and engineering. MFCs are promising devices to address sustainability concerns both in terrestrial and space applications.

Cerium oxide nanoparticles protect cardiac progenitor cells from oxidative stress.

Cardiac progenitor cells (CPCs) are a promising autologous source of cells for cardiac regenerative medicine. However, CPC culture in vitro requires the presence of microenvironmental conditions (a complex array of bioactive substance concentration, mechanostructural factors, and physicochemical factors) closely mimicking the natural cell surrounding in vivo, including the capability to uphold reactive oxygen species (ROS) within physiological levels in vitro. Cerium oxide nanoparticles (nanoceria) are redox-active and could represent a potent tool to control the oxidative stress in isolated CPCs. Here, we report that 24 h exposure to 5, 10, and 50 μg/mL of nanoceria did not affect cell growth and function in cardiac progenitor cells, while being able to protect CPCs from H(2)O(2)-induced cytotoxicity for at least 7 days, indicating that nanoceria in an effective antioxidant. Therefore, these findings confirm the great potential of nanoceria for controlling ROS-induced cell damage.

Multiscale three-dimensional scaffolds for soft tissue engineering via multimodal electrospinning

A novel (scalable) electrospinning process was developed to fabricate bio-inspired multiscale three-dimensional scaffolds endowed with a controlled multimodal distribution of fiber diameters and geared towards soft tissue engineering. The resulting materials finely mingle nano- and microscale fibers together, rather than simply juxtaposing them, as is commonly found in the literature. A detailed proof of concept study was conducted on a simpler bimodal poly(epsilon-caprolactone) (PCL) scaffold with modes of fiber distribution at 600 nm and 3.3 microm. Three conventional unimodal scaffolds with mean diameters of 300 nm and 2.6 and 5.2 microm, respectively, were used as controls to evaluate the new materials. Characterization of the microstructure (i.e. porosity, fiber distribution and pore structure) and mechanical properties (i.e. stiffness, strength and failure mode) indicated that the multimodal scaffold had superior mechanical properties (Youngs modulus approximately 40MPa and strength approximately 1MPa) in comparison with the controls, despite the large porosity ( approximately 90% on average). A biological assessment was conducted with bone marrow stromal cell type (mesenchymal stem cells, mTERT-MSCs). While the new material compared favorably with the controls with respect to cell viability (on the outer surface), it outperformed them in terms of cell colonization within the scaffold. The latter result, which could neither be practically achieved in the controls nor expected based on current models of pore size distribution, demonstrated the greater openness of the pore structure of the bimodal material, which remarkably did not come at the expense of its mechanical properties. Furthermore, nanofibers were seen to form a nanoweb bridging across neighboring microfibers, which boosted cell motility and survival. Lastly, standard adipogenic and osteogenic differentiation tests served to demonstrate that the new scaffold did not hinder the multilineage potential of stem cells.

Characterization of apoptosis signal transduction pathways in HL-5 cardiomyocytes exposed to ischemia/reperfusion oxidative stress model

During ischemia/reperfusion (I/R), cardiomyocytes are exposed to sudden lack of nutrients and successively to radical oxygen species (ROS). In the present study, we used the HL‐5 cardiac atrial myocyte cell line exposed to serum/glucose depletion added or not in H2O2 to mimic ROS during ischemia, then replaced in their standard culture medium to simulate reperfusion. We investigated the effects of serum/glucose depletion combined or not to ROS exposure on AKT and MAP kinases activation to address the role of each event with respect to apoptosis. We demonstrate that serum/glucose depletion per se did not induce apoptosis when compared to ROS exposure. In particular, ROS recruited p38MAPK and JNK pathways. SB202190 preventing p38MAPK activity, partially protected HL‐5 from apoptosis while blocking JNK, thanks to JNKI, further enhanced apoptosis. Blocking phosphatidylinositol (PI) 3‐kinase with LY294002 or ERKs with U0126 was without consequence on apoptosis. Finally, BCL‐2 and BCL‐XL/S expression levels were analyzed in cells exposed to 1 h ischemia followed by 12‐h reperfusion in the presence or not of SB202190; BCL‐2, but not BCL‐XL/S, expression was decreased in ROS treated cells but SB202190 failed to restore BCL‐2 level. Our data suggest that p38MAPK activation primarily mediates ROS‐induced apoptosis while concomitant JNK activation would represent a scavenger pathway for cells trying to escape apoptosis.

Hippo pathway effectors control cardiac progenitor cell fate by acting as dynamic sensors of substrate mechanics and nanostructure

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

Eicosapentaenoic Acid Demethylates a Single CpG That Mediates Expression of Tumor Suppressor CCAAT/Enhancer-binding Protein δ in U937 Leukemia Cells

Polyunsaturated fatty acids (PUFAs) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, α-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFAs affected proliferation; eicosapentaenoic acid (EPA) was the most potent on cell cycle progression. EPA enhanced the expression of the myeloid lineage-specific transcription factors CCAAT/enhancer-binding proteins (C/EBPβ and C/EBPδ), PU.1, and c-Jun, resulting in increased expression of the monocyte lineage-specific target gene, the macrophage colony-stimulating factor receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of macrophage colony-stimulating factor receptor promoter, which is a determinant for expression. We demonstrated that C/EBPβ and C/EBPδ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBPδ, which has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the −370/−20 CpG island on the C/EBPδ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBPδ gene basal expression. Our results provide evidence for a new role of PUFAs in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor C/EBPδ is controlled by the methylation state of a site-specific CpG dinucleotide.

Histatins: salivary peptides with copper(II)- and zinc(II)-binding motifs Perspectives for biomedical applications

Natural antimicrobial peptides represent a primordial mechanism of immunity in both vertebrate and nonvertebrate organisms. Among them, histatins belong to a family of human salivary metal‐binding peptides displaying potent antibacterial, antifungal and wound‐healing activities. These properties, along with the ability of histatins to inhibit collagenases and cysteine proteases, have attracted much attention for their potential use in the treatment of several oral diseases. This review critically assesses the studies carried out to date in order to provide a comprehensive and systematic vision of the information accumulated so far. In particular, the relationship between metal‐binding and peptide activity is extensively analysed. The review provides important clues for developing possible therapeutic applications of histatins and their synthetic peptide analogues by creating a set of necessary resource materials to support investigators and industries interested in exploiting their unique properties.

Human cardiac progenitor cell grafts as unrestricted source of supernumerary cardiac cells in healthy murine hearts.

Human heart harbors a population of resident progenitor cells that can be isolated by stem cell antigen‐1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, less than 1%‐5% survive and differentiate. Among the major causes of this failure are the distressing protocols used to culture in vitro and implant progenitor cells into damaged hearts. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature‐responsive surfaces. In the engineered tissue, progenitor cells established proper three‐dimensional intercellular relationships and were embedded in self‐produced extracellular matrix preserving their phenotype and multipotency in the absence of significant apoptosis. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the murine myocardium and in the vascular walls, where they integrated in the respective textures.

Cooperation of Biological and Mechanical Signals in Cardiac Progenitor Cell Differentiation

Dr. S. Pagliari , Dr. G. Forte , Dr. F. Pagliari , Dr. M. Minieri , Prof. P. Di Nardo Laboratory of Molecular and Cellular Cardiology Department of Internal Medicine University of Rome “Tor Vergata”Rome 00133, Italy E-mail: dinardo@uniroma2.it Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoJapanese-Italian Tissue Engineering Laboratory (JITEL) Tokyo Women’s Medical University-Waseda University Joint Institution for Advanced Biomedical Sciences (TWIns) Tokyo, Japan Dr. S. Pagliari, Dr. G. Forte, Dr. F. Pagliari, Dr. M. Minieri, Prof. P. Di NardoItalian Institute for Cardiovascular Research (INRC) 40126 Bologna, Italy Prof. A. C. Vilela-Silva Instituto de Ciencias Biomedicas and Laboratorio de Tecido Conjuntivo Hospital Universitario Clementino Fraga Filho Rio de Janeiro, Brazil Dr. C. Mandoli , Prof. E. Traversa International Research Center for Materials Nanoarchitectonics (MANA) National Institute for Materials Science (NIMS) 1–1 Namiki, Tsukuba, Ibaraki 305–0044, Japan E-mail: TRAVERSA.Enrico@nims.go.jp Dr. S. Pietronave , Prof. M. Prat Department of Medical Sciences University “A. Avogadro” of Piemonte Orientale 28100 Novara, Italy Dr. G. Vozzi , Prof. A. Ahluwalia Interdepartmental Research Center “E. Piaggio” University of Pisa56126 Pisa, Italy Prof. S. Licoccia , Prof. E. Traversa NAST Centre & Department of Chemical Science and Technology University of Rome “Tor Vergata” Roma 00133, Italy [†] S.P. and A.C.V.S. contributed equally to this work.

Agonist monoclonal antibodies against HGF receptor protect cardiac muscle cells from apoptosis

Hepatocyte growth factor (HGF), a pleiotropic cytokine with mitogenic, motogenic, morphogenic, and antiapoptotic effects in various cell types, is a cardioprotective growth factor that can counteract the loss of cardiomyocytes usually observed in cardiac diseases. HGF is a quite unstable molecule in its biologically active heterodimeric form. Since all HGF-induced biological responses are mediated by its high-affinity tyrosine kinase receptor (Met/HGF-R) encoded by the Met gene, we asked whether a monoclonal antibody (MAb) that displays receptor full agonist activity could protect cardiac muscle cell lines from hydrogen peroxide-induced apoptosis. We report that the MAb efficiently inhibited hydrogen peroxide-induced cell shrinkage, DNA fragmentation, annexin V positivity, mitochondrial translocation of bax, and caspase activation. The MAb was thus able to counteract apoptosis evaluated by both morphological and biochemical criteria. The agonist activity of the MAb was mediated by Met/HGF-R, since a Met/HGF-R-specific short hairpin RNA (shRNA) inhibited both activation of transduction pathways and motility triggered by MAb DO-24. The protective antiapoptotic effect of MAb DO-24 was dependent on activation of the ras-MAPK Erk1/2 and phosphatidylinositol 3-kinase (PI3-kinase)-Akt transduction pathways, since it was abrogated by treatments with their specific pharmacological inhibitors, PD-98059 and wortmannin. Moreover, the MAb induced a motogenic, but not mitogenic, response in these cells, mimicking in all aspects the natural ligand HGF but displaying a significant higher stability than HGF in culture. This MAb may thus be a valuable substitute for HGF, being more easily available in a biologically active, highly stable, and purified form.