Variable endothelial cell function restoration after initiation of two antiretroviral regimens in HIV-infected individuals.

Background: The effect of ART on endothelial cell function is incompletely characterized. Methods: We performed a 24 week prospective, case-control and comparative pilot study of ART-naive HIV-infected patients who started a darunavir- or rilpivirine-based regimen, matched with non-HIV-infected volunteers, to compare changes at week 24 from baseline in levels of circulating endothelial cells (CECs), endothelial progenitor cells (EPCs) and circulating angiogenic cells, as well as changes in immune-activation markers. Results: The study population comprised 24 HIV-infected patients and 24 non-infected volunteers. Both HIV groups completely suppressed viraemia. HIV-infected patients had higher levels of activation markers than the control group in CD8 T cells at baseline; these decreased after 24 weeks of treatment, but without reaching the levels of the control group. No statistical differences in immune activation were seen between the darunavir and rilpivirine groups. Levels of CECs were higher and levels of EPCs and circulating angiogenic cells were lower in HIV-infected patients than in the control group, although these parameters were similar between the darunavir group and the control group, but not the rilpivirine group, at week 24. An unfavourable association was observed between rilpivirine, age and increased number of CECs. Conclusions: Restoration of circulating levels of EPCs and CECs in darunavir-treated patients was greater than in those treated with rilpivirine, suggesting ongoing endothelial repair mechanisms.

Electrical stimulation of cardiac adipose tissue-derived progenitor cells modulates cell phenotype and genetic machinery.

A major challenge of cardiac tissue engineering is directing cells to establish the physiological structure and function of the myocardium being replaced. Our aim was to examine the effect of electrical stimulation on the cardiodifferentiation potential of cardiac adipose tissue-derived progenitor cells (cardiac ATDPCs). Three different electrical stimulation protocols were tested; the selected protocol consisted of 2 ms monophasic square-wave pulses of 50 mV/cm at 1 Hz over 14 days. Cardiac and subcutaneous ATDPCs were grown on biocompatible patterned surfaces. Cardiomyogenic differentiation was examined by real-time PCR and immunocytofluorescence. In cardiac ATDPCs, MEF2A and GATA-4 were significantly upregulated at day 14 after stimulation, while subcutaneous ATDPCs only exhibited increased Cx43 expression. In response to electrical stimulation, cardiac ATDPCs elongated, and both cardiac and subcutaneous ATDPCs became aligned following the linear surface pattern of the construct. Cardiac ATDPC length increased by 11.3%, while subcutaneous ATDPC length diminished by 11.2% (p = 0.013 and p = 0.030 vs unstimulated controls, respectively). Compared to controls, electrostimulated cells became aligned better to the patterned surfaces when the pattern was perpendicular to the electric field (89.71 ± 28.47º for cardiac ATDPCs and 92.15 ± 15.21º for subcutaneous ATDPCs). Electrical stimulation of cardiac ATDPCs caused changes in cell phenotype and genetic machinery, making them more suitable for cardiac regeneration approaches. Thus, it seems advisable to use electrical cell training before delivery as a cell suspension or within engineered tissue.

Optimization of chitosan treatments for managing microflora in lettuce seeds without affecting germination.

Many studies have focused on seed decontamination but no one has been capable of eliminating all pathogenic bacteria. Two objectives were followed. First, to assess the in vitro antimicrobial activity of chitosan against: (a) Escherichia coli O157:H7, (b) native microflora of lettuce and (c) native microflora of lettuce seeds. Second, to evaluate the efficiency of chitosan on reducing microflora on lettuce seeds. The overall goal was to find a combination of contact time and chitosan concentration that reduces the microflora of lettuce seeds, without affecting germination. After treatment lettuce seeds presented no detectable microbial counts (<10(2)CFU/50 seeds) for all populations. Moreover, chitosan eliminated E. coli. Regardless of the reduction in the microbial load, a 90% reduction on germination makes imbibition with chitosan, uneconomical. Subsequent treatments identified the optimal treatment as 10 min contact with a 10 g/L chitosan solution, which maintained the highest germination percentage.

Statistical optimization of a novel low-cost medium based on regional agro-industrial by-products for the production of proteolytic enzymes by Bacillus cereus.

Bacillus sp. are specific producers of peptidase amongst bacteria and peptidase enzymes and are of significant ones due to their multifarious applications. Advances in industrial biotechnology offer potential opportunities for economic utilization of agro-industrial by-products for many biochemical reactions. Due to their rich organic nature, they can serve as an ideal substrate for the production of different value added products like peptidases. In the present work, an attempt was made to optimize different variables by Taguchi methodology for the production of peptidase using agro-industrial by-products hydrolyzed by a Bacillus cereus strain, resulting in brewer's spent grain (BSG) being the optimal organic substrate. Subsequently, operative variables for the BSG were investigated using Taguchi methodology in order to maximize the enzyme production. Additionally, the main medium components were optimized using a mixture design. Finally, the production of peptidase by B. cereus was investigated; also the possible interaction with other proteolytic microbial strains was evaluated. A notorious synergistic effect was observed when B. cereus was inoculated with Pseudomonas sp. These brought a triple benefit, first, opening the possibility to produce technical enzymes at low cost, second, giving greater value to a food industry by-product, and third, reducing the environmental impact caused by the product removal directly into the environment.

Hemosiderin deposits confounds tracking of iron-oxide-labeled stem cells: an experimental study.

BACKGROUND: The aim of the present research was to study the possible interference of hemosiderin deposits with the histological detection of dextran-coated, iron-labeled, mesenchymal stem cells after intracoronary administration in a porcine model of myocardial infarction. MATERIALS AND METHODS: A myocardial infarction was induced in six animals that received intracoronary iron-labeled autologous mesenchymal stem cells (group 1; n = 2) or placebo (group 2; n = 4). Six control animals without myocardial infarction underwent direct intramyocardial injections of iron-labeled autologous mesenchymal stem cells (group 3; n = 2) or placebo (group 4; n = 4). Histological sections from explanted hearts were stained with Prussian blue to identify dextran-coated, iron-labeled, mesenchymal stem cells. RESULTS: After Prussian blue staining, granular blue labeling in the tissue was observed in both groups of animals with infarcts. Similar granular blue labeling was detected in hearts from control animals without infarction that had received iron-labeled mesenchymal stem cells. However, hearts from control animals without infarction that received placebo did not have any granular blue labeling in the tissue. CONCLUSIONS: Hemosiderin from infarction hemorrhage interferes with detection of dextran-coated iron-labeled mesenchymal stem cells after intracoronary administration, suggesting that this marker is not useful to detect mesenchymal stem cells in a porcine model of myocardial infarction.

Postharvest changes in water status and chlorophyll content of lettuce (Lactuca sativa L.) and their relationship with overall visual quality.

The purpose of this study was to evaluate water status, chlorophyll content (C), and overall visual quality (OVQ) of fresh butter lettuce (Lactuca sativa var. Lores) as well as these indexes' evolution during storage and their relationships, if any. Whole lettuce plants were stored at optimal postharvest conditions (0 to 2 degrees C and 97% to 99% relative humidity). Measured parameters during each sampling day were relative water content (RWC), water content (WC), free water (FW), bound water (BW), free water to total water ratio (FW/TW), C, and OVQ. All parameters were evaluated in the external, middle, and internal zones of lettuce heads. The external zone had higher initial values of RWC, WC, and FW than the internal zone. The external zone yielded the highest FW/TW ratio (85%), indicating that external leaves had more water available to be used in degradation reactions and were more perishable, with the lowest shelf life if compared with the other lettuce zones. During storage, water status index evolution differed from zone to zone. An increase in BW and a decrease in FW were detected in all lettuce zones. RWC turned out to be a more sensitive measurement than WC. Yet RWC showed no significant correlation with any index. The OVQ parameter correlates with FW directly, or indirectly through FW/TW in all lettuce zones; therefore, FW is an objective and quantitative measurement, which impacts on the visual quality of butter lettuce. The decrease in chlorophyll content observed in the external leaves strongly correlated with the decrease in OVQ.

Viabilidad celular en un aloinjerto de hueso esponjoso humano criopreservado / Cell viability in a cryopreserved human cancellous allograft

Objetivo. Determinar la persistencia de células vivas en injertos óseos criopreservados y su potencialidad biológica. Material y métodos. Se procesaron diversos fragmentos óseos esponjosos provenientes de un cóndilo femoral crioconservado, de nuestro banco de huesos, utilizado en la cirugía de un recambio de prótesis de cadera. Previo lavado y digestión de los fragmentos, se aislaron células que fueron depositadas en un medio de cultivo alfa-MEM suplementado con un 10% de suero fetal bovino (FBS) (GIBCO) y un 1% de penicilina-estreptomicina (GIBCO) y cultivadas en condiciones estándar a 37° C y 5% CO2 en aire enriquecido (BCO) sin suero. Resultados. Algunas células con aspecto fusiforme se adhirieron al plástico de la placa de cultivo a las 48 horas del procesamiento. Después de 10 días en cultivo, las células empezaron a proliferar rápidamente y a generar colonias. Una vez expandido el cultivo, se procedió al fenotipaje de la población celular aislada. La expresión de los marcadores de superficie analizada mediante citometría de flujo mostró un patrón de expresión similar al obtenido para la población de células madre mesenquimales derivadas de médula ósea humana. Conclusiones. Los aloinjertos óseos criopreservados contienen células viables que pueden corresponder, atendiendo a su estirpe mesenquimal y a su escaso grado de diferenciación morfológica, a precursores de células osteoformadoras

Purpose. To determine the persistence of living cells in cryopreserved bone grafts as well as their biological potential. Materials and methods. Several cancellous bone fragments were processed that had been extracted from a bone-bank cryopreserved femoral condyle used in a revision total hip arthroplasty. After fragment lavage and digestion, a group of cells were isolated that were deposited in an alpha-MEM culture medium supplemented with 10% bovine fetal serum (BFS) (GIBCO) and 1% penicillin-streptomycin (GIBCO) and cultured under standard conditions at serum-free 37° C and 5% CO2 enriched air (BCO). Results. Some spindle-like cells bound to the plastic surface of the culture plate at 48 hs from processing. After 10 days' culture, cells quickly started proliferating and generating colonies. Once the culture was expanded, the isolated population of cells was phenotyped. The expression of the surface markers was analyzed by means of flow cytometry and showed an expression pattern similar to that obtained for the population of mesenchymal stem cells derived from human bone marrow. Conclusions. Bone cryopreserved allografts contain viable cells that could correspond, on the basis of their mesenchymal lineage and their low degree of morphological differentiation, to osteogenerating cell precursors

Dynamics of indigenous microbial populations of butter head lettuce grown in mulch and on bare soil.

The native microflora of lettuce cultivated in mulch and on bare soil and its evolution during storage at optimal condition were evaluated. Inner, mid, and outer leaves of the lettuce heads were analyzed separately and the evolution of the microbial populations were fitted to Gompertz and logistic models. The cultivation method (bare soil and mulch) introduced differences in the initial counts, evolution, and tolerance to refrigeration temperatures for some of the microbial populations under study. Most microbial populations from mulch lettuce presented a decline or little growth under refrigerated storage. However, populations from bare soil lettuce presented some growth phase during storage. Lactic acid bacteria from bare soil lettuce presented significant growth after 8 d of storage while LAB from mulch grown lettuce did not. Concurrently with the LAB growth, there was a decline in the coliform counts in bare soil grown lettuce. At the end of storage, the inner and mid leaves of mulch lettuce presented lower counts of psychrotrophic bacteria, LAB, and yeast and molds.

Umbilical cord blood-derived stem cells spontaneously express cardiomyogenic traits.

BACKGROUND: Umbilical cord blood (UCB) has been widely used for hematopoietic stem cell transplantation. The UCB-derived stem cells (UCBSCs) have been proposed as an alternative to bone marrow (BM)-derived mesenchymal stem cells (MSCs) for cardiac cell-based therapy. Herein we studied whether UCBSCs spontaneously exhibit cardiac-specific markers in vitro. METHODS: Human UCBSCs were isolated, expanded, and phenotyped by flow cytometry, quantitative RT-PCR, and immunofluorescence. Cell pluripotency and proliferation were also assessed by adipogenic and osteogenic media and in growth assays. RESULTS: Among 25 analyzed UCB, 16% of cases afforded primary culture satisfactory generation of UCBSCs. Duplication time (Td) of cultures was 2.16 +/- 0.06 days. The cells were strongly positive for CD105 (18.5 +/- 0.14), CD44 (27 +/- 2.8), CD166 (13 +/- 9), CD29 (59 +/- 9.4), CD90 (60 +/- 11) and consistently negative for CD117 (1.2 +/- 0.1), CD106 (1.1 +/- 0), CD34 (1.2 +/- 0.2), CD14 (1 +/- 0), and CD45 (1 +/- 0), consistent with a mesenchymal lineage. Adipogenesis and osteogenesis of cells resulted in low accumulation of intracellular lipid droplets and high deposition of calcium. The UCBSCs showed gene transcripts for alpha-actinin, connexin (Cx)-43, SERCA-2, and stromal cell-derived factor (SDF)-1alpha. At the protein level, the cells abundantly expressed alpha-actinin, Cx-43, SERCA-2 and SDF-1alpha. In contrast, these cells did not express the cardiac transcription factors GATA-4, Tbx5, and Nkx2.5, nor the sarcomeric proteins beta-myosin heavy chain (beta-MyHC) or cardiac troponin I (cTnI). CONCLUSIONS: Human UCBSCs may represent an alternative source of stem cells for myocardial-cell replacement. These cells can be highly expanded. They spontaneously express proteins of paramount importance for cardiovascular regeneration, such as Cx-43, SERCA-2, and SDF-1alpha.

Identification of cardiomyogenic lineage markers in untreated human bone marrow-derived mesenchymal stem cells.

BACKGROUND: Recent reports refute the classic paradigm by which human heart is unable to repair itself following disease or injury. Cardiac and noncardiac stem cells with cardiac regeneration potential have been documented. We studied whether untreated mesenchymal stem cells express markers of cardiomyogenic lineage in vitro. METHODS: Mesenchymal stem cells were obtained from human iliac crest marrow aspirates. Cells were isolated and characterized using flow cytometry by surface expression of CD105, CD166, CD29, CD44, CD14, and CD34. To evaluate their cardiomyogenic potential, presence of cardiac proteins (cardiac troponin I, sarcomeric alpha-actinin, beta myosin heavy chain (beta-MyHC), connexin-43, and SERCA-2), and transcription factors (GATA-4) were assessed. RESULTS: Mesenchymal stem cells expressed CD105 (4.25 +/- 0.35), CD166 (27.83 +/- 1.89), and CD29 (9.4 +/- 0.57) and were negative for CD34, CD14, and CD45. In absence of additional stimuli in the culture media, these cells expressed connexin-43, alpha-actinin, and GATA-4, and were negative for SERCA-2, cardiac troponin I, and beta-MyHC. CONCLUSIONS: Human adult mesenchymal stem cells spontaneously exhibit markers of cardiac phenotype in vitro. In the appropiate myocardial environment, these cells may transdifferentiate into mature cardiomyocytes.

Regulation of E-cadherin/Catenin association by tyrosine phosphorylation.

Alteration of cadherin-mediated cell-cell adhesion is frequently associated to tyrosine phosphorylation of p120- and beta-catenins. We have examined the role of this modification in these proteins in the control of beta-catenin/E-cadherin binding using in vitro assays with recombinant proteins. Recombinant pp60(c-src) efficiently phosphorylated both catenins in vitro, with stoichiometries of 1.5 and 2.0 mol of phosphate/mol of protein for beta-catenin and p120-catenin, respectively. pp60(c-src) phosphorylation had opposing effects on the affinities of beta-catenin and p120 for the cytosolic domain of E-cadherin; it decreased (in the case of beta-catenin) or increased (for p120) catenin/E-cadherin binding. However, a role for p120-catenin in the modulation of beta-catenin/E-cadherin binding was not observed, since addition of phosphorylated p120-catenin did not modify the affinity of phosphorylated (or unphosphorylated) beta-catenin for E-cadherin. The phosphorylated Tyr residues were identified as Tyr-86 and Tyr-654. Experiments using point mutants in these two residues indicated that, although Tyr-86 was a better substrate for pp60(c-src), only modification of Tyr-654 was relevant for the interaction with E-cadherin. Transient transfections of different mutants demonstrated that Tyr-654 is phosphorylated in conditions in which adherens junctions are disrupted and evidenced that binding of beta-catenin to E-cadherin in vivo is controlled by phosphorylation of beta-catenin Tyr-654.

Independent regulation of adherens and tight junctions by tyrosine phosphorylation in Caco-2 cells.

To study the role of tyrosine phosphorylation in the control of intercellular adhesion of intestinal cells, we have generated several clones of Caco-2 cells that express high levels of pp60v-src only after addition of butyrate. Expression of this oncogene in cells 5 days after confluence induced beta-catenin and p120-ctn tyrosine phosphorylation, redistribution of E-cadherin to the cytosol and disassembly of adherens junctions. However, tight junctions of Caco-2 cells at 5 days after confluence were not altered by expression of pp60v-src. Similar results were obtained when Caco-2 cells were incubated with phosphotyrosine phosphatase inhibitor orthovanadate. Although addition of this compound to postconfluent cells disrupt adherens junctions, tight junctions remain unaltered, as determined measuring monolayer permeability to mannitol or hyperphosphorylation of Triton-insoluble occludin. Modifications in tight junction permeability of Caco-2 were only observed at high concentrations of orthovanadate (1 mM). Interestingly, this tyrosine phosphorylation-refractory state was achieved after confluence since early postconfluent cells (day 2) showed a limited but significant response to low doses of orthovanadate. These results suggest that tight junctions of differentiated Caco-2 cells are uncoupled from adherens junctions and are insensitive to regulation by tyrosine phosphorylation.