Monophasic and Biphasic Electrical Stimulation Induces a Precardiac Differentiation in Progenitor Cells Isolated from Human Heart

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α(1G)) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype

Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

A major obstacle to an effective myocardium stem cell therapy has always been the delivery and survival of implanted stem cells in the heart. Better engraftment can be achieved if cells are administered as cell aggregates, which maintain their extra-cellular matrix (ECM). We have generated spheroid aggregates in less than 24 h by seeding human cardiac progenitor cells (hCPCs) onto methylcellulose hydrogel-coated microwells. Cells within spheroids maintained the expression of stemness/mesenchymal and ECM markers, growth factors and their cognate receptors, cardiac commitment factors, and metalloproteases, as detected by immunofluorescence, q-RT-PCR and immunoarray, and expressed a higher, but regulated, telomerase activity. Compared to cells in monolayers, 3D spheroids secreted also bFGF and showed MMP2 activity. When spheroids were seeded on culture plates, the cells quickly migrated, displaying an increased wound healing ability with or without pharmacological modulation, and reached confluence at a higher rate than cells from conventional monolayers. When spheroids were injected in the heart wall of healthy mice, some cells migrated from the spheroids, engrafted, and remained detectable for at least 1 week after transplantation, while, when the same amount of cells was injected as suspension, no cells were detectable three days after injection. Cells from spheroids displayed the same engraftment capability when they were injected in cardiotoxin-injured myocardium. Our study shows that spherical in vivo ready-to-implant scaffold-less aggregates of hCPCs able to engraft also in the hostile environment of an injured myocardium can be produced with an economic, easy and fast protocol

Correction: Human Cardiac Progenitor Spheroids Exhibit Enhanced Engraftment Potential

[This corrects the article DOI: 10.1371/journal.pone.0137999.]

Site-related airborne biological hazard and seasonal variations in two wastewater treatment plants

Results of a study conducted to assess the degree of airborne bacterial contamination generated by two wastewater treatment plants (WWTP) with different treatment systems and evaluate the dispersion of potential pathogens, have been described. Aerosols samples were collected in summer and winter with an agar impact sampler from several plant sites. External upwind and downwind controls were also examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram- negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to detect pathogenic bacteria. The lowest concentrations of mesophilic and thermophilic bacteria were 8 and 28CFU/m3 in plants A and B respectively, the highest 440000CFU/m3 in both plants. Strains of Escherichia coli, Clostridium perfringens, Staphylococcus aureus and Enterococcus spp. were isolated in some sites of the two plants. Salmonella spp., Yersinia enterocolitica and Legionella spp. were never detected. The activities involving nebulization and mechanical aeration of wastewaters and the sewage inflows have proved to be of greatest potential risk. In both plants, we found a statistically significant dependence of bacterial contamination on the season for many of the analyzed parameters but a clear seasonal trend could not be observed

The assessment of airborne bacterial contamination in three composting plants revealed site-related biological hazard and seasonal variations

Aims:\u2002 The purpose of this study was to evaluate the degree of bacterial contamination generated by three Italian composting plants (1, 2 and 3) in two different seasons and to assess the health risk for the employees. Methods and Results:\u2002 Aerosols samples were collected with an agar impact sampler. Several plant sites and external upwind and downwind controls were examined. Total colony-forming counts of mesophilic and thermophilic bacteria, actinomycetes and streptomycetes, Gram-negatives, coliforms and sulfite-reducers were determined. Selective media were used in order to isolate pathogenic bacteria. The levels of total mesophilic and thermophilic micro-organisms ranged between 33 and >40 000 CFU m 123 in plant 1, 39 and 18 700 CFU m 123 in plant 2 and 261 and 6278 CFU m 123 in plant 3. Strains of Escherichia coli, Staphylococcus aureus and Clostridium perfringens were also found. Conclusions:\u2002 The plants monitored in this study have proved to be sources of aerosolized bacteria. The activities involving mechanical movement of the composting mass and the indoor activities were of greatest potential risk. In all the studied plants, a statistically significant dependence was found between the bacterial contamination and the season for some or almost all the analysed parameters, but a clear seasonal trend could not be observed. Significance and Impact of the Study:\u2002 This study provides broad evidence of bacterial aerosol dispersion and site-related biological hazards that may be useful to the regional government to implement regulations on worker safety in composting plants

Influence of biotic and abiotic factors on human pathogens in a finished compost

The role of indigenous micro\ufb02ora of a \ufb01nished compost, de\ufb01ned NK12, on the growth suppression of pathogens under different moisture and temperature storages was investigated. Total count of mesophilic and thermophilic bacteria was evaluated by the most probable number method and growth of seeded Salmonella arizonae 3924 serogroup B and enteropathogenic Escherichia coli 84M in NK12 at different moisture temperature conditions was monitored. Results on sterile and non-sterile NK12 were compared. In all tested experimental conditions, the NK12 indigenous micro\ufb02ora was stable and biologically active. S. arizonae 3924 and E. coli 84M grew rapidly in sterilized NK12 at different moistures and storage temperatures, and their growth was suppressed in non-sterilized NK12. Pathogens inactivation was lower when compost was stored at 40% and 80% humidity and at 37C. Our results show that the major role in the pathogens suppression was played by the indigenous micro\ufb02ora of the \ufb01nished compost, although physical factors too in\ufb02uenced the growth phenomenon

Diacylglycerol kinases are essential for hepatocyte growth factor-dependent proliferation and motility of Kaposi's sarcoma cells.

Hepatocyte growth factor (HGF) is involved in the pathogenesis of Kaposi's sarcoma (KS), the most frequent neoplasia in patients with AIDS, characterized by proliferating spindle cells, infiltrating inflammatory cells, angiogenesis, edema, and invasiveness. In vitro, this factor sustains the biological behavior of KS derived cells, after activation of its receptor and the downstream MAPK and AKT signals. In other cell types, namely endothelial and epithelial cells, movement, proliferation, and survival stimulated by HGF and other growth factors and cytokines depend on diacylglycerol kinases (DGK). In an effort to identify new intracellular transducers operative in KS cells, which could represent therapeutic targets, we investigated the role of DGK in KS cell movement and proliferation by treating cells with the DGK pharmacological inhibitor R59949. We report that R59949 strongly inhibits HGF-induced KS motility, proliferation, and anchorage-independent growth with only a partial effect on cell adhesion and spreading. R59949 does not affect cell survival, HGF receptor activation, or the classical MAPK and AKT signalling pathways. Furthermore, we carried out an siRNA screen to characterize the DGK isoforms involved in KS motility and anchorage independent growth. Our data indicate a strong involvement of DGK-δ in KS motility and of DGK-ι in anchorage-independent growth. These results indicate that DGK inhibition is sufficient to impair in vitro KS cell proliferation and movement and suggest that selected DGK represent new pharmacological targets to interfere with the malignant properties of KS, independently from the well-known RAS/MAPK and PI3K/AKT pathways