Characterization of Yeast Extracellular Vesicles: Evidence for the Participation of Different Pathways of Cellular Traffic in Vesicle Biogenesis

Background: Extracellular vesicles in yeast cells are involved in the molecular traffic across the cell wall. In yeast pathogens, these vesicles have been implicated in the transport of proteins, lipids, polysaccharide and pigments to the extracellular space. Cellular pathways required for the biogenesis of yeast extracellular vesicles are largely unknown. Methodology/Principal Findings: We characterized extracellular vesicle production in wild type (WT) and mutant strains of the model yeast Saccharomyces cerevisiae using transmission electron microscopy in combination with light scattering analysis, lipid extraction and proteomics. WT cells and mutants with defective expression of Sec4p, a secretory vesicle-associated Rab GTPase essential for Golgi-derived exocytosis, or Snf7p, which is involved in multivesicular body (MVB) formation, were analyzed in parallel. Bilayered vesicles with diameters at the 100-300 nm range were found in extracellular fractions from yeast cultures. Proteomic analysis of vesicular fractions from the cells aforementioned and additional mutants with defects in conventional secretion pathways (sec1-1, fusion of Golgi-derived exocytic vesicles with the plasma membrane; bos1-1, vesicle targeting to the Golgi complex) or MVB functionality (vps23, late endosomal trafficking) revealed a complex and interrelated protein collection. Semi-quantitative analysis of protein abundance revealed that mutations in both MVB- and Golgi-derived pathways affected the composition of yeast extracellular vesicles, but none abrogated vesicle production. Lipid analysis revealed that mutants with defects in Golgi-related components of the secretory pathway had slower vesicle release kinetics, as inferred from intracellular accumulation of sterols and reduced detection of these lipids in vesicle fractions in comparison with WT cells. Conclusions/Significance: Our results suggest that both conventional and unconventional pathways of secretion are required for biogenesis of extracellular vesicles, which demonstrate the complexity of this process in the biology of yeast cells.

On the stabilization of conducting pernigraniline salt by the synthesis and oxidation of polyaniline in hydrophobic ionic liquids

The electrochemical polymerization of aniline in a hydrophobic room-temperature ionic liquid and the spectroelectrochemical characterization of the formed film are presented. The polymerization occurs without the presence of acid in 1-butyl-2,3-dimethylimidazolium bis(trifluoromethanesulfonyl)imide (BMMITFSI), leading to a very stable electroactive material where no degradation was observed even at high applied potentials. Both in situ UV-Vis and Raman spectroscopic studies provided evidence for the stabilization of pernigraniline salt at high oxidation potentials and that this polyaniline state is the conducting form, as was corroborated by in situ resistance measurements. These data are indicative that low conductivity is not an intrinsic property of pernigraniline salt and this point must be reconsidered.

Analysis of Bacteria, Parasites, and Heavy Metals in Lettuce (Lactuca sativa) and Rocket Salad (Eruca sativa L.) Irrigated with Treated Effluent from a Biological Wastewater Treatment Plant

This study aimed to evaluate the viability of using treated residuary water from the Biological Wastewater Treatment Plant of Ribeiro Preto to grow vegetables, through the characterization and quantification of parasites, coliforms, and heavy metals. Three equal cultivation areas were prepared. The first was irrigated with treated/chlorinated (0.2 mg L(-1)) wastewater, the second one with treated wastewater without chlorination, and the third site with potable water, which was the control group. The presence of Hymenolepis nana, Enterobius vermicularis, nematode larvae, and Entamoeba coli was verified in lettuce (Lactuca sativa) samples. Although nematode larvae were observed in rocket salad (Eruca sativa L.), no significant differences were found between the number of parasites and type of irrigation water used. No significant differences were found between the number of fecal coliforms in vegetables and the different types of irrigation. However, the vegetables irrigated with treated effluent without chlorination showed higher levels of fecal coliforms. The risk of pathogens is reduced with bleach addition to the treated effluent at 0.2 mg/L. Concentration of heavy metals in vegetables does not mean significant risks to human health, according with the parameters recommended by the World Health Organization.

Discrete approximation to the global spectrum of the tangent operator for flow past a circular cylinder

This paper deals with the calculation of the discrete approximation to the full spectrum for the tangent operator for the stability problem of the symmetric flow past a circular cylinder. It is also concerned with the localization of the Hopf bifurcation in laminar flow past a cylinder, when the stationary solution loses stability and often becomes periodic in time. The main problem is to determine the critical Reynolds number for which a pair of eigenvalues crosses the imaginary axis. We thus present a divergence-free method, based on a decoupling of the vector of velocities in the saddle-point system from the vector of pressures, allowing the computation of eigenvalues, from which we can deduce the fundamental frequency of the time-periodic solution. The calculation showed that stability is lost through a symmetry-breaking Hopf bifurcation and that the critical Reynolds number is in agreement with the value presented in reported computations. (c) 2007 IMACS. Published by Elsevier B.V. All rights reserved.

Surface Liming and Zinc Availability in a Long-Term Experiment under No-Till System

No-till (NT) system with crop rotation is one of the most effective strategies to improve agricultural sustainability in tropical and subtropical regions. To control soil acidity in NT, lime is broadcast on the surface without incorporation. The increase in soil pH due to surface liming may decrease zinc (Zn) availability and its uptake by crops. A field experiment was performed in Parana State, Brazil, on a loamy, kaolinitic, thermic Typic Hapludox to evaluate Zn bioavailability in a NT system after surface liming and re-liming. Dolomitic lime was surface applied on the main plots in July 1993 at the rates of 0, 2, 4, and 6 Mg ha-1. In June 2000, the main plots were divided in two subplots to study of the effect of surface re-liming at the rates of 0 and 3 Mg ha-1. The cropping sequence was soybean [Glycine max (L.) Merrill] (2001-2 and 2002-3), wheat (Triticum aestivum L.) (2003), soybean (2003-4), corn (Zea mays L.) (2004-5), and soybean (2005-6). Soil samples were collected at the following depths: 0-0.05, 0.05-0.10, and 0.10-0.20m, 10 years after surface liming and 3 years after surface re-liming. Soil Zn levels were extracted by four extractants: (i) 0.005molL-1 diethylenetriaminepentaacetic acid (DTPA) + 0.1molL-1 triethanolamine (TEA) + 0.01molL-1 calcium chloride (CaCl2) solution at pH7.3 (DTPA-TEA), (ii) 0.1molL-1 hydrochloric acid (HCl) solution, (iii) Mehlich 1 solution, and (iv) Mehlich 3 solution. Zinc concentrations in leaves and grains of soybean, wheat, and corn were also determined. Soil pH (0.01molL-1 CaCl2 suspension) varied from 4.4 to 6.1, at the 0- to 0.05-m depth, from 4.2 to 5.3 at the 0.05- to 0.10-m depth, and from 4.2 to 4.8 at the 0.10- to 0.20-m depth, after liming and re-liming. Zinc concentrations evaluated by DTPA-TEA, 0.1molL-1 HCl, Mehlich 1, and Mehlich 3 solutions were not changed as a result of lime rate application. Re-liming increased Zn concentrations extracted by 0.1molL-1 HCl at 0-0.05m deep and by DTPA-TEA at 0.05-0.10m deep. Surface-applied lime promoted a decrease in Zn concentrations of the crops, mainly in grains, because of increased soil pH at the surface layers. Regardless of the liming treatments, levels of Zn were sufficient to soybean, wheat, and corn nutrition under NT.

Effects of refrigeration, freezing and replacement of milk fat by inulin and whey protein concentrate on texture profile and sensory acceptance of synbiotic guava mousses

The effects of refrigeration, freezing and substitution of milk fat by inulin and whey protein concentrate (WPC) on the texture and sensory features of synbiotic guava mousses supplemented with the probiotic, Lactobacillus acidophilus La-5, and the prebiotic fibre oligofructose, were studied. The frozen storage (-18 +/- 1 degrees C), followed by thawing at 4 degrees C before the analyses, and the complete replacement of the milk fat by inulin plus WPC, led to significant differences in the instrumental texture parameters of mousses (P < 0.05). Nonetheless, these changes did not affect the sensory acceptability of the products studied. The frozen storage may be employed to extend the shelf-life of synbiotic guava mousses. Additionally, to obtain a texture profile similar to the traditional product, the simultaneous addition of inulin and WPC is recommended only for the partial replacement of milk fat in refrigerated and frozen mousses, and the total proportion of both ingredients together should not exceed 2.6%. (C) 2010 Elsevier Ltd. All rights reserved.

Viability of Lactobacillus acidophilus in synbiotic guava mousses and its survival under in vitro simulated gastrointestinal conditions

The effects of refrigeration, freezing and substitution of milk fat by inulin and whey protein concentrate (WPC) on Lactobacillus acidophilus La-5 viability and resistance to gastric and enteric simulated conditions in synbiotic guava mousses effects were investigated. Refrigerated mousses supplemented with WPC presented the best probiotic viability. ranging from 7.77 to 6.24 log cfu/g during 28 days of storage. The highest probiotic populations, above 7.45 log cfu/g, were observed for all frozen mousses during 112 days of storage. Decreased L acidophilus survival during the in vitro gastrointestinal simulation was observed both for refrigerated and frozen mousses. Nonetheless, for the refrigerated mousses, the addition of inulin enhanced the probiotic survival during the in vitro assays in the first week of storage. L acidophilus survival in simulated gastrointestinal fluids was also improved through freezing. The frozen storage may be used to provide increased shelf-life for synbiotic guava mousses. Even though the protective effect of inulin and WPC on the probiotic microorganism tested was shown to be more specific for the refrigerated products, the partial replacement of milk fat by these ingredients may also help, as it improves the nutritional value of mousses in both storage conditions. (C) 2009 Elsevier B.V. All rights reserved.

Probiotic potential and sensory properties of coconut flan supplemented with Lactobacillus paracasei and Bifidobacterium lactis

The effect of probiotic cultures on sensory performance of coconut flan during storage at 5 degrees C and the viability of these micro organisms for up to 28 days were investigated. Sensory analyses of the product were performed after 7, 14 and 21 days of storage. Coconut flans were produced with no addition of cultures (T1, control), or supplemented with Bifidobacterium lactis (T2), Lactobacillus paracasei (T3) and B. lactis + L. paracasei (T4). Populations of L. paracasei and B. lactis as single or in co-culture remained above 7 log CFU g(-1) during the entire storage period. Viability of L. paracasei was higher for T3. All products were well accepted and no significant differences (P > 0.05) were detected between the coconut flans studied. The addition of L. paracasei and B. lactis to coconut flan resulted in its having great potential as a functional food, which has high sensory acceptability.

Viability of Lactobacillus acidophilus La-5 added solely or in co-culture with a yoghurt starter culture and implications on physico-chemical and related properties of Minas fresh cheese during storage

The effect of a probiotic culture of Lactobacillus acidophilus (La-5), added solely or in co-culture with a starter culture of Streptococcus thermophilus, on texture, proteolysis and related properties of Minas fresh cheese during storage at 5 degrees C was investigated. Three cheese-making trials were prepared and produced with no addition of cultures (T1 - control), supplemented with La-5 (T2), and with La-5 + S. thermophilus (T3). Viable counts of La-5 remained above 6.00 log cfu g(-1) during the whole storage for T2, reaching 7.00 log cfu g(-1) on the 14th day. For T3, the counts of La-5 remained above 6.00 log cfu g(-1) after 7 days of storage. Due to the presence of S. thermophilus, T3 presented the highest proteolytic index increase and titratable acidity values. Nevertheless, these results and S. thermophilus addition had no influence on viability of La-5 which presented satisfactory populations for a probiotic food. Moreover, the use of a yoghurt culture for the production of Minas fresh cheese T3 supplemented with La-5 resulted in a good quality product, with a small rate of post-acidification, indicating that traditional yoghurt culture could be employed in co-culture with La-5 to improve the quality of this cheese. (C) 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Influence of Lactobacillus paracasei and inulin on instrumental texture and sensory evaluation of fresh cream cheese

The influence of the addition of a potential probiotic culture of Lactobacillus paracasei and of the prebiotic fiber inulin on the texture profile and on the sensory evaluation of probiotic and synbiotic fresh cream-cheeses was monitored. Three cheese-making trials were prepared in quintuplicate, all supplemented with a Streptococcus thermophilus starter culture (T1, T2 and T3). L. paracasei subsp. paracasei was added to T1 and T2, and inulin, to T2. The instrumental texture profile was determined after 1, 7, 14 and 21 days of storage of the cheeses. Sensory evaluation was performed after 7 days of storage. The presence of Lactobacillus paracasei in cheeses T1 and T2 and of inulin in cheeses T2 did not alter the texture profile significantly. Cheeses T1 were the least preferred in the sensory evaluation and differed signifcantly from T2 and T3, due to acidic taste, according to panelists. On the other hand, T2 was the most preferred one, though not significantly different from T3. The addition of the prebiotic ingredient inulin to fresh cream cheese processed with a potentially probiotic Lactobacillus paracasei strain resulted in a product with appropriate features and with aggregated functional properties.

Effect of inulin and Lactobacillus paracasei on sensory and instrumental texture properties of functional chocolate mousse

BACKGROUND: This study evaluated the effect of a potentially probiotic bacteria (Lactobacillus paracasei subsp. paracasei LBC 82), added solely or together with the prebiotic ingredient inulin on instrumental texture attributes and sensory properties of a functional chocolate mousse during storage at 4 +/- 1 degrees C for up to 28 days. RESULTS: The addition of Lactobacillus paracasei resulted in a firmer and more adhesive chocolate mousse. This effect was intensified with the presence of inulin in the synbiotic formulation (5.24 N and -0.956 N, respectively, for firmness and adhesiveness after 28 days of storage) (P < 0.05). L. paracasei population did not vary (P > 0.05) during storage (always between 7.27 and 7.35 log cfu g(-1)), both for the probiotic and the synbiotic mousses. Synbiotic mousse differed from control and probiotic mousses during storage with respect to the color attribute. Moreover, both probiotic and synbiotic mousses presented taste, aroma and texture perceptions which were different from one another and from the control mousse after 14 and 21 days of storage. CONCLUSION: The use of inulin, together with the potentially probiotic strain of Lactobacillus paracasei subsp. paracasei, is advantageous, conferring potentially symbiotic potential to the chocolate mousse, as well as favorable texture and sensory properties. (c) 2008 Society of Chemical Industry.

Sensory evaluation of probiotic Minas fresh cheese with Lactobacillus acidophilus added solely or in co-culture with a thermophilic starter culture

Sensory acceptance of formulations of probiotic Minas fresh cheese was investigated. Cheeses were prepared and supplemented with Lactobacillus acidophilus (T1 - probiotic), Lactobacillus acidophilus + Streptococcus thermophilus (T2 - probiotic + starter) or produced with no addition of cultures (T3 - control). Sensory acceptance tests were performed after 7 and 14 days of storage at 5 degrees C, using a 9-point hedonic scale (1 = dislike extremely; 9 = like extremely). After 7 days, no significant difference was detected among cheeses T1, T2 and T3 (P > 0.05). After 14 days, cheeses T1 and T2 presented higher acceptance and differed significantly from cheeses T3. Cheeses T3 presented significant difference between 7 and 14 days of storage (P < 0.05), whereas probiotic cheeses T1 and T2 were stable in the same period (P > 0.05). The addition of L. acidophilus, either solely or in co-culture with a thermophilic starter culture, resulted in good acceptance of Minas fresh cheese, improving sensory performance of the product during storage.

Inulin and oligofructose improve sensory quality and increase the probiotic viable count in potentially synbiotic petit-suisse cheese

The influence of inulin, oligofructose and oligosaccharides from honey, combined in different proportions, on the consumers` sensory acceptance, probiotic viable count and fructan content of novel potentially synbiotic petit-suisse cheeses was investigated. Probiotic populations varied from 7.20 up to 7.69 log cfu g(-1) (Bifidobacterium animalis subsp. lactis) and from 6.08 up to 6.99 log cfu g(-1) (Lactobacillus acidophilus). The highest fructan contents were achieved by the cheese trials containing oligofructose and/or inulin (above 8.90 g 100 g(-1)). The control trial showed the lowest mean acceptance (6.63) after 28 days of refrigerated storage, whereas the highest acceptance (7.43) was observed for the trial containing 10 g 100 g(-1) oligofructose. Acceptance increased significantly during storage (P < 0.05) only for cheeses supplemented with oligoftuctose and/or inulin. Cheeses containing honey did not perform well enough compared to the cheeses with addition of inulin and/or oligofructose, and the best synbiotic petit-suisse cheese considering sensory and technological functional features was that containing oligofructose and inulin combined, therefore encouraging the commercial product use. (c) 2007 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

In vitro evaluation of gastrointestinal survival of Lactobacillus amylovorus DSM 16698 alone and combined with galactooligosaccharides, milk and/or Bifidobacterium animalis subsp lactis Bb-12

Probiotic properties of Lactobacillus amylovorus DSM 16698 were previously demonstrated in piglets. Here, its potential as a human probiotic was studied in vitro, using the TIM-1 system, which is fully validated to simulate the human upper gastrointestinal tract. To evaluate the effect of the food matrix composition on the survival of L amylovorus DSM 16698 in TIM-1, the microorganism was inoculated alone or with prebiotic galactooligosaccharides (GOS), partially skimmed milk (PSM) and/or commercial probiotic Bifidobacterium animalis subsp. lactis Bb-12 (Bb-12). Samples were collected from TIM-1 for six hours, at one-hour intervals and L amylovorus populations were enumerated on MRS agar plates with confirmation of identity of selected isolates by randomly amplified polymorphic DNA (RAPD) fingerprinting. The cumulative survival for L amylovorus alone (control) was 30% at the end of the experiment (t = 6 h). Co-administration of L amylovorus with GOS. PSM and/or Bb-12 increased its survival in comparison with the control significantly from the 4th hour after ingestion onwards (P<0.05). Furthermore, by the use of High Performance Anion Exchange Chromatography, both L amylovorus and Bb-12 were observed to promptly degrade GOS compounds in samples collected from TIM-1, as assessed at t = 2 h. Hence, food matrix composition interfered with survival and growth of L. amylovorus during passage through TIM-1, providing leads towards optimization of probiotic properties in vivo. (C) 2011 Elsevier B.V. All rights reserved.

Redescription of Riethia truncatocaudata (Edwards, 1931) (Diptera: Chironomidae), with description of female, pupa and larva and generic diagnosis for Riethia

The discovery of the immature life history stages of Riethia truncatocaudata (Edwards, 1931) from South America allows diagnosis of Riethia Kieffer, 1917 in all stages, incorporating reared species from the complete austral range. Pseudochironomus truncatocaudata Edwards, 1931 is a senior synonym (syn. n.) of Pseudochironomus melanoides Edwards, 1931. We redescribe the male to complement the short original descriptions of R. truncatocaudata and R. melanoides. The species is congeneric with Australian Riethia stictoptera Kieffer, the type species of the genus Riethia. Extensive material available from the western Pacific (Australia, New Zealand and New Caledonia) confirms that the diagnosis extends to a gondwanan clade, likely sister group to the largely northern genus Pseudochironomus Kieffer.