The potential use of ozone as antifungal and antiaflatoxigenic agent in nuts and its effect on nutritional quality.

Ozone gas is considered as a safe antimicrobial agent in food industries. Here, we evaluated the antifungal and antiaflatoxigenic activities of ozone against fungal contamination in nuts. The most predominant fungal genera in nuts were Aspergillus, Penicillium, Fusarium, and Rhizopus. Ozone (4 ppm) significantly reduced the fungal sporulation of A. flavus and their aflatoxin production. Interestingly, ozone treatment of nuts reduced the total fungal count and increased aflatoxins degradation by approximately 95% and 85%, respectively. Ozone displayed high efficiency to increase the permeability of cell membrane and injury of cell wall of fungi. Increasing the exposure time of ozone in nuts up to 180 minutes showed to reduce the total lipid, carbohydrates, and protein by around 41.2%, 42.7% and 38.4% respectively, in pistachio, almond and peanuts. In conclusion, ozonation is a suitable decontaminating approach for reducing the microbial load in nuts, when used with suitable exposure time.

Bone regeneration and stem cells.

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.

Transcriptional profiling of myotubes from patients with type 2 diabetes: no evidence for a primary defect in oxidative phosphorylation genes.

AIMS/HYPOTHESIS: Microarray-based studies of skeletal muscle from patients with type 2 diabetes and high-risk individuals have demonstrated that insulin resistance and reduced mitochondrial biogenesis co-exist early in the pathogenesis of type 2 diabetes independently of hyperglycaemia and obesity. It is unknown whether reduced mitochondrial biogenesis or other transcriptional alterations co-exist with impaired insulin responsiveness in primary human muscle cells from patients with type 2 diabetes. METHODS: Using cDNA microarray technology and global pathway analysis with the Gene Map Annotator and Pathway Profiler (GenMapp 2.1) and Gene Set Enrichment Analysis (GSEA 2.0.1), we examined transcript levels in myotubes established from obese patients with type 2 diabetes and matched obese healthy participants, who had been extensively metabolically characterised both in vivo and in vitro. We have previously reported reduced basal lipid oxidation and impaired insulin-stimulated glycogen synthesis and glucose oxidation in these diabetic myotubes. RESULTS: No single gene was differently expressed after correction for multiple testing, and no biological pathway was differently expressed using either method of global pathway analysis. In particular, we found no evidence for differential expression of genes involved in mitochondrial oxidative metabolism. Consistently, there was no difference in mRNA levels of genes known to mediate the transcriptional control of mitochondrial biogenesis (PPARGC1A and NRF1) or in mitochondrial mass between diabetic and control myotubes. CONCLUSIONS/INTERPRETATION: These results support the hypothesis that impaired mitochondrial biogenesis is not a primary defect in the sequence of events leading to insulin resistance and type 2 diabetes.

The histopathology of a human mesenchymal stem cell experimental tumor model: support for an hMSC origin for Ewing's sarcoma?

Sarcomas display varied degrees of karyotypic abnormality, vascularity and mesenchymal differentiation. We have reported that a strain of telomerized adult human bone marrow mesenchymal stem cells (hMSC-TERT20) spontaneously evolved a tumorigenic phenotype after long-term continuous culture. We asked to what extent our hMSC-TERT20 derived tumors reflected events found in human sarcomas using routine histopathological procedures. Early versus late passage hMSC-TERT20 cultures persistently expressed mesenchymal lineage proteins e.g. CD105, CD44, CD99 and vimentin. However, late passage cultures, showed increased immunohistochemical staining for CyclinD1 and p21WAF1/Cip1, whereas p27Kip1 staining was reduced. Notably, spectral karyotyping showed that tumorigenic hMSC-TERT20 cells retained a normal diploid karyotype, with no detectable chromosome abnormalities. Consistent with the bone-forming potential of early passage hMSC-TERT20 cells, tumors derived from late passage cells expressed early biomarkers of osteogenesis. However, hMSC-TERT20 cells were heterogeneous for alpha smooth muscle actin (ASMA) expression and one out of six hMSC-TERT20 derived single cell clones was strongly ASMA positive. Tumors from this ASMA+ clone had distinctive vascular qualities with hot spots of high CD34+ murine endothelial cell density, together with CD34- regions with a branching periodic acid Schiff reaction pattern. Such clone-specific differences in host vascular response provide novel models to explore interactions between mesenchymal stem and endothelial cells. Despite the lack of a characteristic chromosomal translocation, the histomorphology, biomarkers and oncogenic changes were similar to those prevalent for Ewing's sarcomas. The phenotype and ontogenesis of hMSC-TERT20 tumors was consistent with the hypothesis that sarcomas may arise from hMSC, providing a unique diploid model for exploring human sarcoma biology.

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.

Human mesenchymal stem cells: from basic biology to clinical applications.

Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided.

Increased expression of 11beta-hydroxysteroid dehydrogenase type 1 in type 2 diabetic myotubes.

BACKGROUND: Alterations in glucocorticoid hormone metabolism in skeletal muscle have been suggested to contribute to the pathogenesis of the metabolic syndrome. Circulating glucocorticoids consist of inactive cortisone and active cortisol interconverted in various tissues by the enzyme 11beta hydroxysteroid dehydrogenase (HSD). This study aims to investigate whether human myotubes established from healthy obese and matched obese type 2 diabetic (T2D) subjects reveal differences in the expression level of glucocorticoid receptor (GR) and 11beta hydroxysteroid dehydrogenase (HSD1 and HSD2), and to investigate whether chronic exposure to cortisone affects glucose transport. METHODS: In myotubes established from T2D and healthy control subjects we determined the mRNA expression of HSD1, HSD2, GR and determined basal and insulin-stimulated glucose uptake in myotubes precultured with cortisone, cortisol and the HSD1 inhibitor, carbenoxolone for four days. RESULTS: Myotubes established from T2D subjects showed an increased expression of HSD1 mRNA, but with no differences in mRNA of GRalpha, LXRalpha and LXRbeta, whereas HSD2 mRNA was not expressed. Cortisone reduced glucose uptake in diabetic myotubes and the cortisone effect could be abolished by the HSD1 inhibitor carbenoxolone. CONCLUSIONS: Our study shows that cortisone reduces glucose uptake in diabetic myotubes and that this effect seems mediated by an increased mRNA HSD1 expression emphasizing that the local conversion of inactive to active glucocorticoids may be important in the pathogenesis of insulin resistance.

Tissue distribution and engraftment of human mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene.

Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC line (hMSC-TERT). In the present study, we co-transduced hMSC-TERT with enhanced green fluorescent protein gene, and studied tissue distribution, engraftment, and cell survival after intracardiac and intravenous injections in immunodeficient mice. The pattern of organ distribution suggested that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent with several reports on limited systemic transplantability of primary MSC. HMSC-TERT may constitute a valuable tool for mechanistic studies on how to control MSC homing and engraftment.

Glucose does not activate the plasma-membrane-bound H+-ATPase but affects pmaA transcript abundance in Aspergillus nidulans.

The addition of glucose to starved cells of Aspergillus nidulans increased the abundance of the pmaA transcript only transiently (15 min) and to a very low degree (1.3-fold), but strongly decreased its abundance during further incubation. This down-regulation was CreA (carbon catabolite repressor protein)-dependent. Glucose failed to stimulate the plasma membrane (PM)-ATPase activity of A. nidulans, whereas under the same experimental conditions the activity of the enzyme from Saccharomyces cerevisiae was enhanced four-fold within 5-10 min following glucose addition. Glucose stimulated the PM-ATPase of Neurospora crassa only 1.3-fold. Sequence comparison of the C-terminal end of the PM-ATPase from S. cerevisiae, N. crassa, A. nidulans, Fusarium sporotrichoides and Penicillium simplicissimum showed that the two regulatory sites necessary for glucose stimulation in S. cerevisiae are conserved in N. crassa and F. sporotrichoides but not in A. nidulans and P. simplicissimum, and their presence therefore does not correlate with glucose stimulation. We conclude that, in contrast to S. cerevisiae, which has become a paradigm of fungal glucose metabolism, glucose does not up-regulate the activity of the plasma membrane ATPase in the filamentous fungi examined.

The function of CreA, the carbon catabolite repressor of Aspergillus nidulans, is regulated at the transcriptional and post-transcriptional level.

The creA gene of A. nidulans encodes a wide-domain regulatory protein mediating carbon catabolite repression. Northern blot analysis of creA mRNA revealed a complex expression profile: the addition of monosaccharides to a carbon-starved culture of A. nidulans provoked a strong transient stimulation of creA transcript formation within a few minutes. In the case of repressing carbon sources, creA mRNA levels were subsequently downregulated, whereas the high creA mRNA levels were maintained in a creA mutant strain and in the presence of derepressing monosaccharides. A high creA transcript level is essential to achieve carbon catabolite repression and is dependent on glucose transport and, at least partially, on the creB gene product. Subsequent downregulation of creA mRNA levels, on the other hand, is typical of carbon catabolite repression and requires a functional CreA recognition site in the creA promoter (and thus involves autoregulation) and formation of glucose-6-phosphate. Despite the presence of continuing high transcript levels of creA in the presence of derepressing carbohydrates, EMSA demonstrated the presence of only low levels of a CreA-DNA complex in respective cell-free extracts. Upon transfer of carbon catabolite derepressed mycelia to catabolite-repressing conditions, a CreA-DNA complex is formed, and this process is dependent on de novo protein synthesis.