Co-metabolic models of Streptococcus thermophilus in co-culture with Lactobacillus bulgaricus or Lactobacillus acidophilus

To shed light on the interactions occurring in fermented milks when using co-cultures of Streptococcus thermophilus with Lactobacillus bulgaricus (StLb) or Lactobacillus acidophilus (StLa), a new co-metabolic model was proposed and checked either in the presence of Inulin as a prebiotic or not. For this purpose, the experimental data of concentrations of substrates and fermented products were utilized in balances of carbon, reduction degree and ATP. S. thermophilus exhibited always quicker growth compared to the other two microorganisms, while the percentage of lactose fermented to lactic acid, that of galactose metabolized, and the levels of diacetyl and acetoin formed strongly depended on the type of co-culture and the presence of inulin. The StLb co-culture led to higher acetoin and lower diacetyl levels compared to StLa, probably because of more reducing conditions or limited acetoin dehydrogenation. Inulin addition to StLa suppressed acetoin accumulation and hindered that of diacetyl, suggesting catabolite repression of alpha-acetolactate synthase expression in S. thermophilus. Both co-cultures showed the highest ATP requirements for biomass growth and maintenance at the beginning of fermentation, consistently with the high energy demand of enzyme induction during lag phase. Inulin reduced these requirements making biomass synthesis and maintenance less energy-consuming. Only a fraction of galactose was released from lactose, consistently with the galactose-positive phenotype of most dairy strains. The galactose fraction metabolized without inulin was about twice that in its presence, which suggests inhibition of the galactose transport system of S. thermophilus by fructose released from partial inulin hydrolysis. (C) 2012 Elsevier B.V. All rights reserved.

Involvement of formyl peptide receptors in the stimulatory effect of crotoxin on macrophages co-cultivated with tumour cells

Crotoxin (CTX) is the main neurotoxic component of Crotalus durissus terrificus snake venom. It inhibits tumour growth and modulates the function of macrophages, which are essential cells in the tumour microenvironment. The present study investigated the effect of CTX on the secretory activity of monocultured macrophages and macrophages co-cultivated with LLC-WRC 256 cells. The effect of the macrophage secretory activities on tumour cell proliferation was also evaluated. Macrophages pre-treated with CTX (0.3 μg/mL) for 2 h were co-cultivated with LLC-WRC 256 cells, and the secretory activity of the macrophages was determined after 12, 24 and 48 h. The co-cultivation of CTX-treated macrophages with the tumour cells caused a 20% reduction in tumour cell proliferation. The production of both H2O2 and NO was increased by 41% and 29% after 24 or 48 h of co-cultivation, respectively, compared to the values for the co-cultures of macrophages of control. The level of secreted IL-1β increased by 3.7- and 3.2-fold after 12 h and 24 h of co-cultivation, respectively. Moreover, an increased level of LXA4 (25%) was observed after 24 h of co-cultivation, and a 2.3- and 2.1-fold increased level of 15-epi-LXA4 was observed after 24 h and 48 h, respectively. Boc-2, a selective antagonist of formyl peptide receptors, blocked both the stimulatory effect of CTX on the macrophage secretory activity and the inhibitory effect of these cells on tumour cell proliferation. Taken together, these results indicate that CTX enhanced the secretory activity of macrophages, which may contribute to the antitumour activity of these cells, and that activation of formyl peptide receptors appears to play a major role in this effect.

Stem cells from umbilical cord blood do have myogenic potential, with and without differentiation induction in vitro

The dystrophin gene, located at Xp21, codifies dystrophin, which is part of a protein complex responsible for the membrane stability of muscle cells. Its absence on muscle causes Duchenne Muscular Dystrophy (DMD), a severe disorder, while a defect of muscle dystrophin causes Becker Muscular Dystrophy (DMB), a milder disease. The replacement of the defective muscle through stem cells transplantation is a possible future treatment for these patients. Our objective was to analyze the potential of CD34+ stem cells from umbilical cord blood to differentiate in muscle cells and express dystrophin, in vitro. Protein expression was analyzed by Immunofluorescence, Western Blotting (WB) and Reverse Transcriptase – Polymerase Chain Reaction (RT-PCR). CD34+ stem cells and myoblasts from a DMD affected patient started to fuse with muscle cells immediately after co-cultures establishment. Differentiation in mature myotubes was observed after 15 days and dystrophin-positive regions were detected through Immunofluorescence analysis. However, WB or RT-PCR analysis did not detect the presence of normal dystrophin in co-cultures of CD34+ and DMD or DMB affected patients' muscle cells. In contrast, some CD34+ stem cells differentiated in dystrophin producers' muscle cells, what was observed by WB, reinforcing that this progenitor cell has the potential to originate muscle dystrophin in vitro, and not just in vivo like reported before.

Altered phenotype and function of dendritic cells in individuals with chronic periodontitis

OBJECTIVE: To investigate the effects of periodontal bacterial lysates on maturation and function of mature monocyte-derived dendritic cells (m-MDDCs) derived from individuals with chronic periodontitis (CP) or healthy periodontal tissue (HP). DESIGN: m-MDDCs derived from peripheral blood monocytes, cultured for 7 days in presence of interleukin (IL)-4 and granulocyte-macrophage colony stimulating factor (GM-CSF), were stimulated with lysates of Streptococcus sanguinis, Prevotella intermedia, Porphyromonas gingivalis, or Treponema denticola on day 4, and were then phenotyped. IL-10, IL-12 and IFN-gamma concentration in the supernatant of cultures were measured. RESULTS: Expression of HLA-DR was lower in bacterial-unstimulated mature m-MDDC from CP compared to HP (p=0.04), while expression of CD1a and CD123 were higher in CP. The expression pattern of HLA-DR, CD11c, CD123, and CD1a did not change on bacterial stimulation, regardless of the bacteria. Stimulation with P. intermedia upregulated CD80 and CD86 in CP cells (p≤0.05). Production of IL-12p70 by bacterial-unstimulated m-MDDCs was 5.8-fold greater in CP compared to HP. Bacterial stimulation further increased IL-12p70 production while decreasing IL-10. Significantly more IFN-gamma was produced in co-cultures of CP m-MDDCs than with HP m-MDDCs when cells were stimulated with P. intermedia (p=0.009). CONCLUSIONS: Bacterial-unstimulated m-MDDC from CP exhibited a more immature phenotype but a cytokine profile biased towards proinflammatory response; this pattern was maintained/exacerbated after bacterial stimulation. P. intermedia upregulated co-stimulatory molecules and IFN-gamma expression in CP m-MDDC. These events might contribute to periodontitis pathogenesis

The cytolethal distending toxin of Aggregatibacter actinomycetemcomitans inhibits macrophage phagocytosis and subverts cytokine production

Aggregatibacter actinomycetemcomitans is an important periodontal pathogen that can participate in periodontitis and other non-oral infections. The cytolethal distending toxin (Cdt) is among the virulence factors produced by this bacterium. The Cdt is also secreted by several mucosa-associated Gram-negative pathogens and may play a role in perpetuating the infection by modulating the immune response. Although the toxin targets a wide range of eukaryotic cell types little is known about its activity on macrophages which play a key part in alerting the rest of the immune system to the presence of pathogens and their virulence factors. In view of this, we tested the hypothesis that the A. actinomycetemcomitans Cdt (AaCdt) disrupts macrophage function by inhibiting phagocytic activity as well as affecting the production of cytokines. Murine macrophages were co-cultured with either wild-type A. actinomycetemcomitans or a Cdt(-) mutant. Viable counts and qPCR showed that phagocytosis of the wild-type strain was significantly reduced relative to that of the Cdt(-) mutant. Addition of recombinant Aa(r)Cdt to co-cultures along with the Cdt(-) mutant diminished the phagocytic activity similar to that observed with the wild type strain. High concentrations of Aa(r)Cdt resulted in decreased phagocytosis of fluorescent bioparticles. Nitric oxide production was modulated by the presence of Cdt and the levels of IL-1β, IL-12 and IL-10 were increased. Production of TNF-α did not differ in the co-culture assays but was increased by the presence of Aa(r)Cdt. These data suggest that the Cdt may modulate macrophage function in A. actinomycetemcomitans infected sites by impairing phagocytosis and modifying the pro-inflammatory/anti-inflammatory cytokine balance.

Growth, organic acids profile and sugar metabolism of Bifidobacterium lactis in co-culture with Streptococcus thermophilus: the inulin effect

The organic acids profile, sugar metabolism and biomass growth of Streptococcus thermophilus (St) and Bifidobacterium lactis (BI) have been studied in pure cultures or binary co-culture (St-BI) in skim milk either containing 40 mg/g of inulin or not. With inulin, the time required by St. BI and St-BI to complete fermentation (i.e., when the pH reached 4.5) was about 14, 8 and 49% shorter than without inulin, respectively. This prebiotic also enhanced the levels of lactic and acetic acids and volatile compounds, showing a positive synbiotic effect between pre- and probiotics. In particular, the St-BI co-culture showed final concentrations of both microorganisms about 15 and 38% higher than in their respective pure cultures, thus highlighting a clear synergistic effect between these microorganisms due to mutual interactions. In addition, the well-known bifidogenic effect of inulin was confirmed. (c) 2012 Elsevier Ltd. All rights reserved.

Sardinian goat's milk as source of bacteriocinogenic potential protective cultures

Goat breeding in Sardinia constitutes an important source of income for farming and shepherding activities. In this study 170 LAB strains were isolated from Sardinian goat's milk and tested for bacteriocins production against several food-borne pathogenic microorganisms. Four isolates (SD1, SD2, SD3 and SD4) were selected for their effective inhibition on Listeria monocytogenes. The strains were classified as members of Enterococcus genus, according to their biochemical and physiological characteristics, and then genetically identified as Enterococcus faecium. In MRS broth at 37 degrees C, bacteriocins SD1 and SD2 were produced at much higher levels (51200 AU/ml) compared to bacteriocin SD3 (3200 AU/ml) and bacteriocin SD4 (800 AU/ml). Their peptides were inactivated by proteolytic enzymes, but not when treated with alpha-amylase, catalase and lipase. The four bacteriocins remained stable at pH from 2.0 to 12.0, after exposure to 100 degrees C for 120 min and were not affected by the presence of surfactants and salts (N-Laourylsarcosine, NaCl, SDS, Triton X-100, Tween 20, Tween 80 and urea). Their molecular size was determined to be approximately 5 kDa by tricine-SDS-PAGE. Since the strains exhibited a strong antimicrobial activity against 21 L monocytogenes strains and 6 Salmonella spp. isolates, they should be considered as potential bio-preservatives cultures for fermented food productions. Moreover, due to their technological features, the four strains could be taken in account for using as adjunct NSLAB (non-starter lactic acid bacteria) rather than as starter culture. (C) 2011 Elsevier Ltd. All rights reserved.

Combustion of coal, bagasse and blends thereof Part I: Emissions from batch combustion of fixed beds of fuels

Batch combustion of fixed beds of coal, bagasse and blends thereof took place in a pre-heated two-stage electric laboratory furnace, under high-heating rates. The average input fuel/air equivalence ratios were similar for all fuels. The primary and secondary furnace temperatures were varied from 800 degrees C to 1000 degrees C. The effects of fuel blending, combustion staging, and operating furnace temperatures on the emissions from the two fuels were assessed. Furnace effluents were analyzed for carbon dioxide and for products of incomplete combustion (PIC) including CO, volatile and semi-volatile hydrocarbons, as well as particulate matter. Results showed that whereas CO2 was generated during both the observed sequential volatile matter and char combustion phases of the fuels, PICs were only generated during the volatile matter combustion phase. CO2 emissions were the highest from coal, whereas CO and other PIC emissions were the highest from bagasse. Under this particular combustion configuration, combustion of the volatile matter of the blends resulted in lower yields of PIC, than combustion of the volatiles of the neat fuels. Though CO and unburned hydrocarbons from coal as well as from the blends did not exhibit a clear trend with furnace temperature, such emissions from bagasse clearly increased with temperature. The presence of the secondary furnace (afterburner) typically reduced PIC, by promoting further oxidation of the primary furnace effluents. (C) 2012 Elsevier Ltd. All rights reserved.

SYNTHESIS OF N-15-ENRICHED UREA (CO((NH2)-N-15)(2)) FROM (NH3)-N-15, CO, AND S IN A DISCONTINUOUS PROCESS

CO((NH2)-N-15)(2) enriched with the stable isotope N-15 was synthesized based on a reaction involving CO, (NH3)-N-15, and S in the presence of CH3OH. The method differs from the industrial method; a stainless steel reactor internally lined with polytetrafluoroethylene (PTFE) was used in a discontinuous process under low pressure and temperature. The yield of the synthesis was evaluated as a function of the parameters: the amount of reagents, reaction time, addition of H2S, liquid solution and reaction temperature. The results showed that under optimum conditions (1.36, 4.01, and 4.48 g of (NH3)-N-15, CO, and S, respectively, 40 ml CH3OH, 40 mg H2S, 100 degrees C and 120 min of reaction) 1.82 g (yield 76.5%) of the compound was obtained per batch. The synthesized CO((NH2)-N-15)(2) contained 46.2% N, 0.55% biuret, melting point of 132.55 degrees C and did not exhibit isotopic fractionation. The production cost of CO((NH2)-N-15)(2) with 90.0 at. % N-15 was US$ 238.60 per gram.

Effects of photobioreactor configuration, nitrogen source and light intensity on the fed-batch cultivation of Arthrospira (Spirulina) platensis. Bioenergetic aspects

Bioenergetic analysis may be applied in order to predict microbial growth yields, based on the Gibbs energy dissipation and mass conservation principles of the overall growth reaction. The bioenergetics of the photoautotrophic growth of the cyanobacterium Arthrospira (Spirulina) platensis was investigated in different bioreactor configurations (tubular photobioreactor and open ponds) using different nitrogen sources (nitrate and urea) and under different light intensity conditions to determine the best growing conditions in terms of Gibbs energy dissipation, number of photons to sustain cell growth and phototrophic energy yields distribution in relation to the ATP and NADPH formation, and release of heat. Although an increase in the light intensity increased the Gibbs energy dissipated for cell growth and maintenance with both nitrogen sources, it did not exert any appreciable influence on the moles of photons absorbed by the system to produce one C-mol biomass. On the other hand, both bioenergetic parameters were higher in cultures with nitrate than with urea, likely because of the higher energy requirements needed to reduce the former nitrogen source to ammonia. They appreciably increased also when open ponds were substituted by the tubular photobioreactor, where a more efficient light distribution ensured a remarkably higher cell mass concentration. The estimated percentages of the energy absorbed by the cell showed that, compared with nitrate, the use of urea as nitrogen source allowed the system to address higher energy fractions to ATP production and light fixation by the photosynthetic apparatus, as well as a lower fraction released as heat. The best energy yields values on Gibbs energy necessary for cell growth and maintenance were achieved in up to 4-5 days of cultivation, indicating that it would be the optimum range to maintain cell growth. Thanks to this better bioenergetic situation, urea appears to be a quite promising low-cost, alternative nitrogen source for Arthrospira platensis cultures in photobioreactors. (C) 2011 Elsevier Ltd. All rights reserved.

Effect of inulin on the growth and metabolism of a probiotic strain of Lactobacillus rhamnosus in co-culture with Streptococcus thermophilus

Metabolic studies are very important to improve quality of functional dairy products. For this purpose, the behaviors of pure cultures of Streptococcus thermophilus (St) and Lactobacillus rhamnosus (Lr) as well a co-culture of them (St-Lr) were investigated during skim milk fermentation, and the inulin effect as prebiotic was assessed. Lr was able to metabolize 6 g/100 g more galactose than St and St-Lr. Final lactic acid production by Lr was higher (9.8 g/L) compared to St (9.1 g/L) and St-Lr (9.1 g/L). Acetic acid concentration varied from 0.8 g/L (St-Lr) to 1.5 g/L (Lr) and that of ethanol from only 0.2 g/L (St-Lr) to 0.4 g/L (Lr), which suggests the occurrence in Lr of a NADH oxidase activity and citrate co-metabolization via pyruvate, both dissipating a part of the reducing power. Diacetyl and acetoin accumulated at the highest levels (18.4 and 0.8 mg/L, respectively) with St-Lr, which suggests possible synergistic interactions between these microorganisms as well as the Lr capability of co-metabolizing citrate in the presence of lactose. Inulin stimulated both biomass growth and levels of all end-products, as the likely result of fructose release from its partial hydrolysis and subsequent metabolization as an additional carbon and energy source. Crown Copyright (C) 2012 Published by Elsevier Ltd. All rights reserved.

Synthesis of 15N-enriched urea (CO(15NH2)2) from 15NH3, CO, and S in a discontinuous process

CO(15NH2)2 enriched with the stable isotope 15N was synthesized based on a reaction involving CO, 15NH3, and S in the presence of CH3OH. The method differs from the industrial method; a stainless steel reactor internally lined with polytetrafluoroethylene (PTFE) was used in a discontinuous process under low pressure and temperature. The yield of the synthesis was evaluated as a function of the parameters: the amount of reagents, reaction time, addition of H2S, liquid solution and reaction temperature. The results showed that under optimum conditions (1.36, 4.01, and 4.48 g of 15NH3, CO, and S, respectively, 40 ml CH3OH, 40 mg H2S, 100 ºC and 120 min of reaction) 1.82 g (yield 76.5%) of the compound was obtained per batch. The synthesized CO(15NH2)2 contained 46.2% N, 0.55% biuret, melting point of 132.55 ºC and did not exhibit isotopic fractionation. The production cost of CO(15NH2)2 with 90.0 at. % 15N was US$ 238.60 per gram.