Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production

Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (similar to 2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

Evaluation of the role of environmental factors in the human gastrointestinal tract on the behaviour of probiotic cultures of Lactobacillus casei Shirota and Lactobacillus casei LC01 by the use of a semi-dynamic in vitro model

This study evaluated the influence of gastrointestinal environmental factors (pH, digestive enzymes, food components, medicaments) on the survival of Lactobacillus casei Shirota and Lactobacillus casei LC01, using a semi-dynamic in vitro model that simulates the transit of microorganisms through the human GIT. The strains were first exposed to different simulated gastric juices for different periods of time (0, 30, 60 and 120 min), and then to simulated intestinal fluids for zero, 120, 180 and 240 min, in a step-wise format. The number of viable cells was determined after each step. The influence of food residues (skim milk) in the fluids and resistance to medicaments commonly used for varied therapeutic purposes (analgesics, antiarrhythmics, antibiotics, antihistaminics, proton pump inhibitors, etc.) were also evaluated. Results indicated that survival of both cultures was pH and time dependent, and digestive enzymes had little influence. Milk components presented a protective effect, and medicaments, especially anti-inflammatory drugs, influenced markedly the viability of the probiotic cultures, indicating that the beneficial effects of the two probiotic cultures to health are dependent of environmental factors encountered in the human gastrointestinal tract.

Antimicrobial compounds produced by Lactobacillus sakei subsp sakei 2a, a bacteriocinogenic strain isolated from a Brazilian meat product

Bacteriocins produced by lactic acid bacteria are gaining increased importance due to their activity against undesirable microorganisms in foods. In this study, a concentrated acid extract of a culture of Lactobacillus sakei subsp. sakei 2a, a bacteriocinogenic strain isolated from a Brazilian pork product, was purified by cation exchange and reversed-phase chromatographic methods. The amino acid sequences of the active antimicrobial compounds determined by Edman degradation were compared to known protein sequences using the BLAST-P software. Three different antimicrobial compounds were obtained, P1, P2 and P3, and mass spectrometry indicated molecular masses of 4.4, 6.8 and 9.5 kDa, respectively. P1 corresponds to classical sakacin P, P2 is identical to the 30S ribosomal protein S21 of L. sakei subsp. sakei 23 K, and P3 is identical to a histone-like DNA-binding protein HV produced by L. sakei subsp. sakei 23 K. Total genomic DNA was extracted and used as target DNA for PCR amplification of the genes sak, lis and his involved in the synthesis of P1, P2 and P3. The fragments were cloned in pET28b expression vector and the resulting plasmids transformed in E. coli KRX competent cells. The transformants were active against Listeria monocytogenes, indicating that the activity of the classical sakacin P produced by L. sakei 2a can be complemented by other antimicrobial proteins.

Evaluation of culture media for enumeration of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium animalis in the presence of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus

The study compared the growth capability of probiotic (Lactobacillus acidophilus La05, Lactobacillus casei Lc01 and Bifidobacterium animalis Bb12) and non-probiotic (Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus) cultures on twenty-one culture media grouped according to selectivity: nonselective agars, selective agars without antibiotics and MRS agars containing different combinations of lithium chloride, cystein, bile salts and antibiotics. Four of these media were selected for quantitative enumeration of L acidophilus La05, L casei Lc01, and B. animalis Bb12. The best culture media and incubation conditions for enumeration of the probiotic cultures were: B. animalis: MRS agar with dicloxacillin, 37 degrees C or 42 degrees C, anaerobiosis; L acidophilus: MRS agar with bile salts, 37 degrees C or 42 degrees C, aerobiosis; L casei: MRS agar with lithium chloride and sodium propionate, 37 degrees C or 42 degrees C, aerobiosis or anaerobiosis. Plating on MRS with glucose replaced by maltose, 37 degrees C or 42 degrees C, anaerobiosis, will distinguish probiotic from non-probiotic cultures. For enumeration of each probiotic in a mixed culture, the following media and incubation conditions were recommended: B. animalis: 4ABC-MRS, 42 degrees C, anaerobiosis, L acidophilus: LC medium, 42 degrees C, aerobiosis or anaerobiosis and L casei: LP-MRS, 42 degrees C, aerobiosis or anaerobiosis. In all experiments, differences in counts using pour plating or surface plating were not significant (P <= 0.05). (C) 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Entecavir Treatment for up to 5 Years in Patients with Hepatitis B e Antigen-Positive Chronic Hepatitis B

Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of 183 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-901. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received >= 1 year of entecavir 0.5 mg in ETV-022 and then entered ETV-901 with a treatment gap <= 35 days. In ETV-901 the entecavir dose was 1.0 mg daily. For patients with samples available at Year 5, proportions with hepatitis B virus (HBV) DNA <300 copies/mL, normal alanine aminotransferase (ALT) levels, HBeAg loss, and HBeAg seroconversion were determined. In all, 146 patients met criteria for inclusion in the entecavir long-term cohort. At Year 5, 94% (88/94) had HBV DNA <300 copies/mL and 80% (78/98) had normal ALT levels. In addition to patients who achieved serologic responses during study ETV-022, 23% (33/141) achieved HBeAg seroconversion and 1.4% (2/145) lost hepatitis B surface antigen (HBsAg) during study ETV-901. Through 5 years, entecavir resistance emerged in one patient. The safety profile of entecavir was consistent with previous reports. Conclusion: Extended therapy with entecavir through 5 years maintained or increased rates of HBV DNA suppression and ALT normalization. Additional patients also achieved HBeAg loss and seroconversion. Entecavir provides sustained viral suppression with minimal resistance during long-term treatment of HBeAg-positive CHB. (HEPATOLOGY 2010;51:422-430.)

Climatological features of cutoff low systems in the Southern Hemisphere

Cutoff lows (COLs) pressure systems climatology for the Southern Hemisphere (SH), between 10 degrees S and 50 degrees S, using the National Center for Environmental Prediction-National Center for Atmospheric Research (NCEP-NCAR) and the ERA-40 European Centre for Medium Range Weather Forecast (ECMWF) reanalyses are analyzed for the period 1979-1999. COLs were identified at three pressure levels (200, 300, and 500 hPa) using an objective method that considers the main physical characteristics of the conceptual model of COLs. Independently of the pressure level analyzed, the climatology from the ERA-40 reanalysis has more COLs systems than the NCEP-NCAR. However, both reanalyses present a large frequency of COLs at 300 hPa, followed by 500 and 200 hPa. The seasonality of COLs differs at each pressure level, but it is similar between the reanalyses. COLs are more frequent during summer, autumn, and winter at 200, 300, and 500 hPa, respectively. At these levels, they tend to occur around the continents, preferentially from southeastern Australia to New Zealand, the south of South America, and the south of Africa. To study the COLs at 200 and 300 hPa from a regional perspective, the SH was divided in three regions: Australia-New Zealand (60 E-130 W), South America (130 degrees W-20 degrees W), and southern Africa (20 degrees W-60 degrees E). The common COLs features in these sectors for both reanalyses are a short lifetime (similar to 80.0% and similar to 70.0% of COLs at 200 and 300 hPa, respectively, persisting for up to 3 days), mobility (similar to 70.0% and similar to 50% of COLs at 200 and 300 hPa, respectively, traveling distances of up to 1200 km), and an eastward propagation.

Integration of Ligand- and Target-Based Virtual Screening for the Discovery of Cruzain Inhibitors

A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.