73 research outputs found
Accurate and unambiguous tag-to-gene mapping in serial analysis of gene expression
BACKGROUND: In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. RESULTS: We applied this method to the Saccharomyces cerevisiae genome, producing the most thorough and accurate annotation of potential virtual SAGE tags that is available today for this organism. The usefulness of this method is exemplified by the significant reduction of ambiguous cases in existing experimental SAGE data. In addition, we report new insights from the analysis of existing SAGE data. First, we found that experimental SAGE tags mapping onto introns, intron-exon boundaries, and non-coding RNA elements are observed in all available SAGE data. Second, a significant fraction of experimental SAGE tags was found to map onto genomic regions currently annotated as intergenic. Third, a significant number of existing experimental SAGE tags for yeast has been derived from truncated cDNAs, which are synthesized through oligo-d(T) priming to internal poly-(A) regions during reverse transcription. CONCLUSION: We conclude that an accurate and unambiguous tag mapping process is essential to increase the quality and the amount of information that can be extracted from SAGE experiments. This is supported by the results obtained here and also by the large impact that the erroneous interpretation of these data could have on downstream applications
Sustainable production of β-Xanthophylls in Saccharomyces Cerevisiae
Xanthophylls are a group of C40 pigments that belong to the carotenoids family. β-Xanthophylls, such as zeaxanthin, violaxanthin and neoxanthin are derived from β-carotene metabolism, and play a central role in the protection of photo-oxidative damage in plants and algae. These molecules have interesting applications as precursors of commercially relevant natural aromas, like safranal and damascenone. Furthermore, zeaxanthin is also widely used as a nutraceutical to improve ocular health. In this study, we engineered the yeast Saccharomyces cerevisiae to biosynthesize zeaxanthin and violaxanthin from glucose. We used integrative vectors to construct a genetic stable β-xanthophylls pathway in a β-carotenogenic yeast strain. To find an effective zeaxanthin biosynthetic enzyme, we compared the titers achieved by bacterial, plant and algal β-carotene hydroxylases. Additionally, we evaluated the effect of the chloroplast transit peptide of plant and algal enzymes on zeaxanthin biosynthesis. The strain that expressed truncated version of Solanum lycopersicum β-carotene hydroxylase showed the best performance, reaching up to 4.7 mg/g DCW of zeaxanthin after 72 h cultivation in shake-flasks. Zeaxanthin producing strains were transformed with zeaxanthin epoxidase genes to further extend the pathway to violaxanthin, which was measured by UPLC-MS. To the best of our knowledge, this work presents the highest titer of zeaxanthin in S. cerevisiae reported to date, the first zeaxanthin cell factory using β-carotene hydroxylase from plants, and the first heterologous biosynthesis of violaxanthin.
Financial support of FONDECYT grant No.1170745 is greatly acknowledged. Vicente F. Cataldo acknowledges CONICYT for receiving graduate scholarship
The Implementation of Payroll Accounting System as an Effort to Improve the System of Internal Control (a Study at Pdam Kota Malang)
This research aims to determine the implementation of payroll accounting system in PDAM Kota Malang as an effort to improve the internal control system and to provide the alternative problem solving if obstacles related to the system exists. Using descriptive method, the researcher obtained relevant data by using interview and documentation. The result shows that the system is well-implemented and it supports the internal control. It is evidenced by the involvement of related functions in the processing the payroll effectively. Also, the authorization of every job is clear and following the procedure. This study suggests the company to add more specific information related to income tax discounted from the employees\u27 take home pays. This research also recommends the company to apply more complete flowchart of payroll system
Contextualized genome-scale model unveils high-order metabolic effects of the specific growth rate and oxygenation level in recombinant Pichia pastoris
Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/g per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris
Foreign Direct Investment and Economic Growth in Developing Countries: How Relevant Are Host-Country and Industry Characteristics?
Development of an International Odor Identification Test for Children: The Universal Sniff Test
Objective: To assess olfactory function in children and to create and validate an odor identification test to diagnose olfactory dysfunction in children, which we called the Universal Sniff (U-Sniff) test. Study design: This is a multicenter study involving 19 countries. The U-Sniff test was developed in 3 phases including 1760 children age 5-7 years. Phase 1: identification of potentially recognizable odors; phase 2: selection of odorants for the odor identification test; and phase 3: evaluation of the test and acquisition of normative data. Test—retest reliability was evaluated in a subgroup of children (n = 27), and the test was validated using children with congenital anosmia (n = 14). Results: Twelve odors were familiar to children and, therefore, included in the U-Sniff test. Children scored a mean ± SD of 9.88 ± 1.80 points out of 12. Normative data was obtained and reported for each country. The U-Sniff test demonstrated a high test—retest reliability (r27 = 0.83, P < .001) and enabled discrimination between normosmia and children with congenital anosmia with a sensitivity of 100% and specificity of 86%. Conclusions: The U-Sniff is a valid and reliable method of testing olfaction in children and can be used internationally
Production of β‑ionone by combined expression of carotenogenic and plant CCD1 genes in Saccharomyces cerevisiae
Background
Apocarotenoids, like the C13-norisoprenoids, are natural compounds that contribute to the flavor and/or aroma of flowers and foods. They are produced in aromatic plantslike raspberries and rosesby the enzymatic cleavage of carotenes. Due to their pleasant aroma and flavour, apocarotenoids have high commercial value for the cosmetic and food industry, but currently their production is mainly assured by chemical synthesis. In the present study, a Saccharomyces cerevisiae strain that synthesizes the apocarotenoid -ionone was constructed by combining integrative vectors and high copy number episomal vectors, in an engineered strain that accumulates FPP.
Results
Integration of an extra copy of the geranylgeranyl diphosphate synthase gene (BTS1), together with the carotenogenic genes crtYB and crtI from the ascomycete Xanthophyllomyces dendrorhous, resulted in carotenoid producing cells. The additional integration of the carotenoid cleavage dioxygenase gene from the plant Petunia hybrida (PhCCD1) let to the production of low amounts of -ionone (0.073 ± 0.01 mg/g DCW) and changed the color of the strain from orange to yellow. The expression of the crtYB gene from a high copy number plasmid in this former strain increased -ionone concentration fivefold (0.34 ± 0.06 mg/g DCW). Additionally, the episomal expression of crtYB together with the PhCCD1 gene in the same vector resulted in a final 8.5-fold increase of -ionone concentration (0.63 ± 0.02 mg/g DCW). Batch fermentations with this strain resulted in a final specific concentration of 1 mg/g DCW at 50 h, which represents a 15-fold increase.
Conclusions
An efficient -ionone producing yeast platform was constructed by combining integrative and episomal constructs. By combined expression of the genes BTS1, the carotenogenic crtYB, crtI genes and the plant PhCCD1 genethe highest -ionone concentration reported to date by a cell factory was achieved. This microbial cell factory represents a starting point for flavor production by a sustainable and efficient process that could replace current methods.This work was funded by grants COPEC-UC 6C-063 and FONDECYT No 1130822, and the Novo Nordisk Foundation
Productive development policies in Latin America: past and present.
This paper reviews industrial policy in Latin America from the Great Depression to our
days. Its purpose is to derive some lessons for what Latin American and Caribbean
countries (LAC) should do in this area. It has become clear over the last few years that
LAC, if they are to accelerate their growth rates, need more than a good macroeconomic
framework and the protection of property rights: they need to be more proactive in
transforming their production structures, still too dependent on primary commodity exports
or the assembly of final goods from imported components, sectors that are ill-suited to the
productive development jumps that have been associated with high growth in the
developing world over the past 60 years
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