How yeast cells deal with stalled replication forks.

DNA polymerases sometimes stall during DNA replication at sites where DNA is damaged, or upon encounter with proteins or secondary structures of DNA. When that happens, the polymerase clamp PCNA can become modified with a single ubiquitin moiety at lysine 164, opening DNA Damage Tolerance (DDT) mechanisms that either repair or bypass the lesions. An alternative repair mechanism is the salvage recombination (SR) pathway, which copies information from the sister chromatid. SUMOylation of PCNA at the same lysine, or at lysine 127, can recruit the Srs2 helicase, which negatively controls SR. Recently, we have dissected the relationship between SR and the DDT pathways, and showed that overexpression of either the PCNA unloader Elg1, or the Rad52 homologous recombination protein, can bypass the repression by Srs2. Our results shed light on the interactions between different DNA damage repair/bypass proteins, and underscore the importance of PCNA modifications in organizing the complex task of dealing with DNA damage during replication of the genetic material.

Saccharomyces cerevisiae from Brazilian kefir-fermented milk: An in vitro evaluation of probiotic properties.

The therapeutic use of probiotics for supporting the antibiotic action against gastrointestinal disorders is a current trend and emerging applications have gained popularity because of their support for various microbiological activities in digestive processes. Microorganisms isolated from kefir with great probiotic properties, in addition to high resistance to harsh environmental conditions, have been widely researched. Administration of probiotic yeasts offers a number of advantages, when compared to bacteria, because of particular characteristics as their larger cell size. In the present study, 28 strains of Saccharomyces cerevisiae were isolated, after in vitro digestion of kefir-fermented milk, and identified by molecular based approaches. A screening was performed to determine important quality requirements for probiotics including: antagonistic and antioxidant activities, ß-galactosidase synthesis, autoaggregation, surface hydrophobicity and adhesion to epithelial cells. The results showed strains: with antagonistic activity against microbial pathogens such as Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis; able to produce ß-galactosidase; with antioxidant activity levels higher than 90%; with hydrophobicity activity and autoaggregation ability (evaluated by adhesion test, where all the strains presented adhesion to mice ileal epithelial cells). These findings are relevant and the strains are recommended for further in vivo studies as well as for potential therapeutic applications.

Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays.

We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine.

Assessment of the efficacy of the feed additive consisting of Saccharomyces cerevisaeCNCM I-1077 (Levucell®SC) for dairy cows, cattle for fattening, minor ruminant species and camelids (Lallemand SAS).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the re-evaluation of the authorisation of the feed additive containing Saccharomyces cerevisae CNCM I-1077 (Levucell® SC) as a zootechnical additive for ruminants. The additive is already authorised for use in dairy sheep and dairy goats, lambs and horses, calves, all minor ruminant species (for rearing) other than lambs and camelids (for rearing) and dairy cows. The additive is intended for use in complete feed for dairy cows, minor ruminant species for milk production and all camelids at the minimum dose of 4 × 108 CFU/kg and for cattle for fattening and minor ruminant species for fattening at the minimum dose of 5 × 108 CFU/kg. In a previous opinion, the FEEDAP Panel concluded that the additive has a potential to improve the performance of cattle raised for fattening, minor ruminant species and camelids raised for meat production but could not conclude on the efficacy of Levucell® SC for dairy cows or extrapolate to minor dairy ruminant species or dairy camelids. The applicant has provided supplementary information to support efficacy of the additive in dairy cows. Based on the data provided, the FEEDAP Panel is not in the position to conclude on the efficacy of the additive for dairy cows or other dairy ruminants under the proposed conditions of use.

Taraxacum brevicorniculatum rubber elongation factor TbREF associates with lipid droplets and affects lipid turn-over in yeast.

A protein named TbREF that is localized on rubber particles of the rubber producing dandelion species Taraxacum brevicorniculatum was expressed in tobacco leaves and in yeast. TbREF fused to fluorescence proteins colocalized on globular, hydrophobic structures, most likely lipid droplets. Furthermore, triacylglycerol, sterol and total lipid content of TbREF expressing yeast was determined by photometric analyses of nile red stainings and GC-MS analyses. Therefore, yeast exposed an enhanced nile red fluorescence as well as an increased TAG and sterol content compared to wildtype and vector control. Altogether, these findings gave new insights into the putative function of TbREF that might be pushing rubber particle production due to its cytotoxic nature and/or shielding and preventing degradation of lipid droplets. Furthermore, these results highlight possible biotechnological applications regarding the accumulation of hydrophobic compounds in lipid droplet like structures.

A fine-tuned composition of protein nanofibrils yields an upgraded functionality of displayed antibody binding domains.

Elevated performance of instruments and electronic devices is frequently attained through miniaturization of the involved components, which increases the number of functional units in a given volume. Analogously, to conquer the limitations of materials used for the purification of monoclonal antibodies and for the sensitivity of immunoassays, the support for capturing antibodies requires miniaturization. A suitable scaffold for this purpose are cross-ß structured protein nanofibrils, as they offer a superior surface area over volume ratio and because manipulation can be implemented genetically. To display the antibody binding Z-domain dimers (ZZ) along the surface of the fibrils and grant maximal accessibility to the functional units, the N-terminal fragments of the fibrillating translation release factor Sup35 or ureidosuccinate transporter Ure2, both from Saccharomyces cerevisae, are simultaneously fibrillated with the chimeric-proteins Sup35-ZZ and ZZ-Ure2, respectively. Optimization of the fibril composition yields a binding capacity of 1.8 mg antibody per mg fibril, which is a binding capacity that is almost 20-fold higher, compared to the commercially available affinity medium gold standard, protein A sepharose. This study lifts the craft of nanofibril functionalization to the next level, and offers a universal framework to improve biomaterials that rely on the display of functional proteins or enzymes.

Cryptic 3' mRNA processing signals hinder the expression of Schistosoma mansoni integrins in yeast.

The expression of parasite genes has often proven difficult in heterologous systems such as yeast or E. coli. Most often, promoter choice and codon usage were hypothesised to be the main reason for expression failures. The trematode parasite Schistosoma mansoni has five integrin genes named Smα-Int1-4 and Smß-Int1, which we aimed to express in the yeast Saccharomyces cerevisiae. This has not been achieved, however, as only Smß-Int1 integrin could be expressed. When the four α integrins were driven by a stronger promoter, this enabled Smα-Int1 to be expressed as well, but the remaining integrins, Smα-Int2-4, still could not be expressed. Evidence from RT-PCR experiments suggested that this was due to premature transcription termination. Using detailed in silico sequence analyses we identified AT-rich stretches in these integrin genes, which have high similarity to yeast mRNA 3'-end processing signals. We hypothesised that these signals were causing the premature truncation. To test this, we designed an optimised version of Smα-Int3, in which the sequence was modified to replace the yeast 3' processing signals. This strategy allowed us to express Smα-Int3 integrin successfully in S. cerevisiae. These findings show that the misinterpretation of AT-rich sequences by yeast 3'-mRNA processing machinery can cause problems when attempting to express genes containing such sequences in this host.

The ADH7 Promoter of Saccharomyces cerevisiae is Vanillin-Inducible and Enables mRNA Translation Under Severe Vanillin Stress.

Vanillin is one of the major phenolic aldehyde compounds derived from lignocellulosic biomass and acts as a potent fermentation inhibitor to repress the growth and fermentative ability of yeast. Vanillin can be reduced to its less toxic form, vanillyl alcohol, by the yeast NADPH-dependent medium chain alcohol dehydrogenases, Adh6 and Adh7. However, there is little information available regarding the regulation of their gene expression upon severe vanillin stress, which has been shown to repress the bulk translation activity in yeast cells. Therefore, in this study, we investigated expression patterns of the ADH6 and ADH7 genes in the presence of high concentrations of vanillin. We found that although both genes were transcriptionally upregulated by vanillin stress, they showed different protein expression patterns in response to vanillin. Expression of Adh6 was constitutive and gradually decreased under vanillin stress, whereas expression of Adh7 was inducible, and, importantly, occurred under severe vanillin stress. The null mutants of ADH6 or ADH7 genes were hypersensitive to vanillin and reduced vanillin less efficiently than the wild type, confirming the importance of Adh6 and Adh7 in vanillin detoxification. Additionally, we demonstrate that the ADH7 promoter is vanillin-inducible and enables effective protein synthesis even under severe vanillin stress, and it may be useful for the improvement of vanillin-tolerance and biofuel production efficiency via modification of yeast gene expression in the presence of high concentrations of vanillin.

Antifungal activities of the essential oil and its fractions rich in sesquiterpenes from leaves of Casearia sylvestris Sw

ABSTRACT Casearia genus (Salicaceae) is found in sub-tropical and tropical regions of the world and comprises about 160-200 species. It is a medicinal plant used in South America, also known as "guaçatonga", "erva-de-tiú", "cafezinho-do-mato". In Brazil, there are about 48 species and 12 are registered in the State of Rio de Janeiro, including Casearia sylvestris Sw. There are many studies related to the chemical profile and cytotoxic activities of extracts from these plants, although few studies about the antifungal potential of the essential oil have been reported. In this work, we have studied the antifungal properties of the essential oil of C. sylvestris leaves, as well as of their fractions, against four yeasts (Saccharomyces cerevisae, Candida albicans, C. glabrata and C. krusei) for the first time. The chemical analysis of the essential oil revealed a very diversified (n = 21 compounds) volatile fraction composed mainly of non-oxygenated sesquiterpenes (72.1%). These sesquiterpenes included α-humulene (17.8%) and α-copaene (8.5%) and the oxygenated sesquiterpene spathulenol (11.8%) were also identified. Monoterpenes were not identified. The fractions are mainly composed of oxygenated sesquiterpenes, and the most active fraction is rich in the sesquiterpene 14-hydroxy -9-epi-β-caryophyllene. This fraction was the most effective in inhibiting the growth of three yeast strains.

Hematological and biochemical responses of pirarucu (Arapaima gigas, Arapaimidae) fed with diets containing a glucomannan product derived from yeast and algae / Respostas hematológicas e bioquímicas de pirarucu (Arapaima gigas, Arapaimidae) alimentado com dietas contendo mananoligossacarídeos derivados de leveduras e algas

ABSTRACT The hematological and biochemical responses of pirarucu fingerlings (Arapaima gigas) fed with diets containing different concentrations of a glucomannan product derived from yeast and algae were evaluated in order to ascertain the effect of these diets on fish physiology. Four treatments were conducted, with three replications, with 12 fish in each tank. The product evaluated (MycosorbA+(r)) was incorporated into the commercial diet, at four concentrations: 0, 1, 2 and 4 g.kg-1, called M0%, M0.1%, M0.2% and M0.4%, respectively. After 45 days of feeding, blood samples from six fish in each replicate were collected to perform the analyses. Their weight and length were determined to calculate the condition factor and weight gain, but no differences (P > 0.05) were observed among the treatments. No changes to the hematocrit, hemoglobin or erythrocyte levels or to the hematimetric indices of the pirarucus were observed. The glucose and triglyceride levels of the pirarucus in the M0.1% and M0.2% groups were significantly lower than those of the M0% group. The M0.2% group showed higher albumin levels (P < 0.05) than M0% and M0.4%. The M0.4% group showed a total cholesterol level that was significantly higher than in all other treatments. MycosorbA+(r) contributed towards increasing the levels of defense cells in A. gigas. It would be possible to use this product at concentrations of between 0.1% and 0.2%, given that they increase the levels of some defense cells and plasma albumin concentrations, without changes to hematological parameters, cholesterol and triglyceride plasma levels or condition factor.

RESUMO As características hematológicas e bioquímicas de alevinos de pirarucu Arapaima gigas alimentados com diferentes concentrações de mananoligossacarídeos derivados de leveduras e algas na dieta foram avaliadas para verificar o efeito sobre sua fisiologia. Quatro tratamentos foram conduzidos com três repetições (12 peixes em cada). O produto avaliado (MycosorbA+(r)) foi incorporado à ração comercial, em quatro níveis: 0, 1, 2 e 4 g kg-1 de ração, denominados M0%, M0,1%, M0,2% e M0,4%, respectivamente. Após 45 dias de alimentação, amostras de sangue de seis peixes de cada repetição foram coletadas para realização das análises. O peso e comprimento foram obtidos para cálculo do fator de condição e ganho de peso, entretanto, não foram observadas diferenças (P>0,05) entre os tratamentos. Assim como não foram observadas alterações nos valores de hematócrito, hemoglobina, eritrócitos e índices hematimétricos dos pirarucus. A concentração de glicose e triglicérides dos pirarucus dos grupos M0,1% e M0,2% foram significativamente menores que o tratamento M0%. Os peixes do tratamento M0,2% apresentaram níveis de albumina maior (P<0,05) que do M0% e M0,4%. Os peixes alimentados com M0,4% mostraram nível de colesterol total significativamente maior que todos os demais tratamentos. MycosorbA+(r) contribuiu para o aumento das células de defesa de A. gigas, podendo ser utilizado nas concentrações de 0,1% a 0,2%, devido ao aumento do número de certas células de defesa e dos níveis plasmáticos de albumina e por não ter sido observadas outras alterações nos parâmetros hematológicos, nos níveis plasmáticos de colesterol e triglicérides e no fator de condição.

Desempenho e características bioquímicas de leitões submetidos a dietas com aditivos probióticos, prebióticos, simbióticos e antibióticos / Performance and biochemical parameters of piglets and the use of alternative additives as antibiotic replacement

Avaliaram-se o desempenho e as características bioquímicas de leitões desmamados, utilizando-se aditivos alternativos em substituição aos antibióticos. Foram utilizados 480 leitões machos, distribuídos em delineamento inteiramente ao acaso, composto por cinco tratamentos. A ração foi à base de milho, farelo de soja e um núcleo comercial para leitões. Os tratamentos consistiram em T1: ração sem aditivos, T2: dieta basal com antibiótico, T3: dieta basal com adição de prebiótico, T4: dieta basal com adição de probiótico e T5: dieta basal com adição de simbiótico. Foram avaliados peso corporal, ganho de peso, consumo de ração e conversão alimentar. Em 50 amostras de sangue de leitões, sendo 10 amostras de cada tratamento, avaliaram-se as concentrações séricas de proteínas totais, de albumina e globulina, a relação albumina:globulina e a fosfatase alcalina, creatinina, alanina aminotransferase, aspartato aminotransferase, gama- glutamiltransferase, colesterol, ureia, cálcio e fósforo e a relação cálcio:fósforo. A substituição de antibióticos por aditivos alternativos não influenciou o desempenho nem as características bioquímicas dos leitões.

The experiment was conducted to evaluate the performance and biochemical parameters in weaned piglets using alternative supplements to replace antibiotics. A total of 480 male pigs were used, randomly distributed in delineation with five treatments. The feed was based on corn, soybean meal and a commercial core for piglets. The treatments were T1: diet without supplements or basal diet, T2: basal diet with antibiotics, T3: basal diet with added probiotic, T4: basal diet with added probiotic and T5: basal diet with addition of symbiotic. In the experiment we evaluated performance characteristics, body weight, weight gain, feed intake and feed conversion. In the second part of the experiment, we evaluated the biochemical parameters of piglets used in the first experiment, fifty blood samples, ten samples of each treatment, were taken, and after separation of the clot, the serum was frozen at -20°C. Subsequently we analyzed the serum levels of total protein, albumin, globulin, albumin/globulin, ALP, creatinine, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, cholesterol, urea, calcium, phosphorus and calcium/phosphorus ratio. No significant differences in biochemical parameters or in the performance of piglets submitted to different diets were found.