Biomedical subjects

Jens Nielsen

Publications and source records attributed to Jens Nielsen.

At least 19 recordsLinked to original sources

PubMed2007

A systems biology approach to study glucose repression in the yeast Saccharomyces cerevisiae.

Glucose repression in the yeast Saccharomyces cerevisiae has evolved as a complex regulatory system involving several different pathways. There are two main pathways involved in signal transduction. One has a role in glucose sensing and regulation of glucose transport, while another takes part in repression of a wide range of genes involved in utilization of alternative carbon sources. In this work, we applied a systems biology approach to study the interaction between these two pathways. Through genome-wide transcription analysis of strains with disruption of HXK2, GRR1, MIG1, the combination of MIG1 and MIG2, and the parental strain, we identified 393 genes to have significantly changed expression levels. To identify co-regulation patterns in the different strains we applied principal component analysis. Disruption of either GRR1 or HXK2 were both found to have profound effects on transcription of genes related to TCA cycle and respiration, as well as ATP synthesis coupled proton transport, all displaying an increased expression. The hxk2Delta strain showed reduced overflow metabolism towards ethanol relative to the parental strain. We also used a genome-scale metabolic model to identify reporter metabolites, and found that there is a high degree of consistency between the identified reporter metabolites and the physiological effects observed in the different mutants. Our systems biology approach points to close interaction between the two pathways, and our metabolism driven analysis of transcription data may find a wider application for analysis of cross-talk between different pathways involved in regulation of metabolism.

Gene Expression Profiling↗

PubMed2006

Transcription analysis using high-density micro-arrays of Aspergillus nidulans wild-type and creA mutant during growth on glucose or ethanol.

Here, we describe how the recently published Aspergillus nidulans genome sequence [Galagan, J.E., Calvo, S.E., Cuomo, C., Li-Jun, M., Wortman, J.R., et al., 2005. Sequencing of Aspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438 (7071), 1105-1115] was used to design a high-density oligo array with probes for 3,278 selected genes using the Febit Geniom One array system. For this purpose, the program OligoWiz II was used to design 24,125 probes to cover the 3,278 selected genes. Subsequently, the Febit system was used to investigate carbon catabolite repression by comparing the gene expression of a creA deleted mutant strain with a reference strain grown either with glucose or ethanol as the sole carbon source. In order to identify co-regulated genes and genes influenced by either the carbon source or CreA, the most significantly regulated genes (p<or=0.01) were grouped in eight clusters based on their expression profile. Analysis of the clusters allowed identification of numerous genes that are presumably not regulated by CreA, or alternatively are either directly or indirectly regulated by CreA. Surprisingly, we found evidence that more than 25% of the genes (54 out of the 200 significantly regulated) that are repressed by glucose are not completely de-repressed during growth on ethanol, as deletion of the creA resulted in increased expression of the genes in question even during growth on ethanol. Thus, the expression profiles obtained in the eight clusters indicate that the carbon catabolite repression is not a simple on/off switch but a more complex system not only dependent on the presence or absence of CreA but also on the carbon source.

Aspergillus nidulans↗

PubMed2006

Community cohort study of rotavirus and other enteropathogens: are routine vaccinations associated with sex-differential incidence rates?

OBJECTIVE: Community studies in West Africa have demonstrated that routine vaccinations may have non-targeted effects, the female-male mortality ratio being reduced after administration of BCG and increased after diphtheria-tetanus-pertussis (DTP). We examined whether immunisation status was associated with infection with rotavirus and other enteropathogens. METHODS: We recruited 200 children shortly after birth and followed them until 2 years of age with weekly morbidity interviews and stool sampling. Vaccination status for each child was classified according to the most recent vaccination as documented by vaccination card. MAIN OUTCOME MEASURES: The female-male incidence rate ratios (IRR) of infection with an enteropathogen and of enteropathogen-associated diarrhoea were estimated for children according to whether they had received BCG or DTP as their last vaccination. RESULTS: For children who received BCG as their last vaccine, the adjusted female-male IRRs for primary rotavirus-infection and diarrhoea were 1.05 (95% CI: 0.21-5.28) and 0.0 (95% CI: 0-3.02), respectively. For children who received DTP as their last vaccine, the adjusted female-male IRRs were 1.93 (0.89-4.21) and 1.92 (0.70-5.32), respectively, for rotavirus-associated infection and diarrhoea. Restricted to the rotavirus season, the female-male IRRs for rotavirus infection and diarrhoea were 2.56 (1.17-5.63) and 2.63 (0.94-7.34), respectively. The female-male IRR for rotavirus-associated diarrhoea differed significantly among BCG and DTP recipients (p=0.02). Infections with enteropathogens not associated with diarrhoea were associated with lower female-male IRRs after BCG of 0.82 (0.55-1.23) and higher female-male IRRs after DTP vaccination of 1.32 (1.03-1.70) for primary infection (p=0.05). Though there were few infections with other diarrhoea-causing enteropathogens, these were also associated with a lower female-male IRR after BCG of 0.62 (0.26-1.52) and a higher female-male IRR after DTP vaccination of 1.51 (1.04-2.20) for all infection. CONCLUSION: Routine immunisations may affect morbidity for non-targeted infections. As in studies of infant mortality, BCG is associated with lower risk for girls, whereas, DTP is associated with higher risk for girls relative to boys.

BCG Vaccine↗

PubMed2006

[Reduced post-partum bleeding after treatment with sublingual misoprostol: a randomized double-blind clinical study in a developing country--secondary publication)].

Post-partum haemorrhage is the most common cause of maternal death in third-world countries. The objective of this study was to evaluate whether routine administration of sublingual misoprostol, 600 g, reduces post-partum haemorrhage. Significantly fewer women in the misoprostol group experienced a loss of > or = 1,000 ml (11% vs. 17%; 0.66 (0.45-0.98)) or > or = 1,500 ml (2% vs. 8%; 0.28 (0.12-0.64)) of blood. We conclude that sublingual misoprostol reduces the frequency of severe post-partum haemorrhage.

Administration, Sublingual↗

PubMed2006

Hap4 is not essential for activation of respiration at low specific growth rates in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the heme-activated protein complex Hap2/3/4/5 plays a major role in the transcription of genes involved in respiration. Thus, overexpression of HAP4 has been shown to result in a 10% increase in the respiratory capacity. Here the physiology of a HAP4-deleted S. cerevisiae strain was investigated, and we found that the hap4delta S. cerevisiae exhibited poor growth on ethanol, although the growth rate on glucose was indifferent from the wild type in aerobic as well as anaerobic cultures. Moreover, it exhibited a large (75%) reduction in the critical glucose uptake rate at which fermentative metabolism is onset, indicating a substantial reduction in respiratory capacity. We also performed whole genome transcription analysis for the hap4delta and the wild type, grown in carbon-limited chemostat cultures operated at a dilution rate of 0.05 h(-1). Although both strains exhibited respiratory metabolism, there was significant change in expression of many genes in the hap4delta strain. These genes are involved in several different parts of the metabolism, including oxidative stress response, peroxisomal functions, and energy generation. This study strongly indicates that Hap4 activation only occurs at intermediate specific growth rates, below which the transcription of genes responsible for respiration is dependent on the Hap2/3/5 complex and above which the Hap4 protein augments the transcription. Furthermore, statistical analysis of the transcription data and integration of the data with a genome scale metabolic network provided new insight and evidence for the role of Hap4 in transcriptional regulation of mitochondrial respiration.

Biomass↗

PubMed2006

Fungal metabolite analysis in genomics and phenomics.

Metabolomics consists of strategies to quantitatively identify cellular metabolites and to understand how trafficking of these biochemical messengers through the metabolic network influences phenotype. The application of metabolomics to fungi has been strongly pursued because these organisms are widely used for the production of chemicals, are well known for their diverse metabolic landscape and serve as excellent eukaryotic model organisms for studying metabolism and systems biology. Within the context of fungal systems, recent progress has been made in the development of analytical tools and mathematical strategies used in metabolite analysis that have enhanced our ability to crack the code underpinning the cellular inventory, regulatory schemes and communication mechanisms that dictate cellular function. Metabolomics has played a key role in functional genomics and strain classification.

Fungi↗

PubMed2006

Community cohort study of Cryptosporidium parvum infections: sex-differential incidences associated with BCG and diphtheria-tetanus-pertussis vaccinations.

OBJECTIVE: Community studies in West Africa have suggested that routine vaccinations may have sex-differential non-targeted effects, the female-male mortality ratios being increased after receiving diphtheria-tetanus-pertussis (DTP) vaccination and reduced after administration of BCG or measles vaccine (MV). Using an existing data set, we examined whether vaccinations were associated with gender-differential incidences of Cryptosporidium parvum infection. METHODS: Two hundred children had been recruited shortly after birth and followed until 2 years of age or until follow-up was interrupted by a war. We performed weekly morbidity interviews and collected stool specimens, irrespective of whether the children had diarrhoea. Vaccination status for each child was classified according to the most recent vaccination with BCG, DTP, or MV. FINDINGS: The female-male incidence rate ratio (IRR) for Cryptosporidium infection among children who had received BCG as their last vaccine was 0.0 (95% CI: 0-3.49). However, among those who had received DTP as their last vaccine, the female-male IRR was 6.25 (2.06-18.9) for Cryptosporidium infection and 3.60 (0.91-14.2) for Cryptosporidium-associated diarrhoea. The female-male IRRs for Cryptosporidium infection differed significantly among BCG and DTP recipients (p=0.01). Among children who had received measles as their last routine vaccine, the female-male IRR was 1.57 (0.60-4.11) for Cryptosporidium infection and 0.98 (0.28-3.52) for Cryptosporidium-associated diarrhoea. The female-male IRRs for Cryptosporidium infection differed among DTP and MV recipients (p=0.02). For girls, early DTP vaccination compared with late or no DTP vaccination was associated with increased incidence rate of Cryptosporidium infection (IRR=4.23 (1.04-17.2)). For girls, the incidence rate decreased when they received MV. INTERPRETATION: Routine immunisations may affect morbidity for non-targeted infections. As in studies of infant mortality, BCG is associated with a low risk for girls relative to boys, whereas DTP is associated with a high female-male IRR of C. parvum infection.

Animals↗

PubMed2006

Correlation between the presence of neutralizing antibodies against porcine circovirus 2 (PCV2) and protection against replication of the virus and development of PCV2-associated disease.

BACKGROUND: In a previous study, it was demonstrated that high replication of Porcine circovirus 2 (PCV2) in a gnotobiotic pig was correlated with the absence of PCV2-neutralizing antibodies. The aim of the present study was to investigate if this correlation could also be found in SPF pigs in which PMWS was experimentally reproduced and in naturally PMWS-affected pigs. RESULTS: When looking at the total anti-PCV2 antibody titres, PMWS-affected and healthy animals seroconverted at the same time point, and titres in PMWS-affected animals were only slightly lower compared to those in healthy animals. In healthy animals, the evolution of PCV2-neutralizing antibodies coincided with that of total antibodies. In PMWS-affected animals, neutralizing antibodies could either not be found (sera from field studies) or were detected in low titres between 7 and 14 DPI only (sera from experimentally inoculated SPF pigs). Differences were also found in the evolution of specific antibody isotypes titres against PCV2. In healthy pigs, IgM antibodies persisted until the end of the study, whereas in PMWS-affected pigs they quickly decreased or remained present at low titres. The mean titres of other antibody isotypes (IgG1, IgG2 and IgA), were slightly lower in PMWS-affected pigs compared to their healthy group mates at the end of each study. CONCLUSION: This study describes important differences in the development of the humoral immune response between pigs that get subclinically infected with PCV2 and pigs that experience a high level of PCV2-replication which in 3 of 4 experiments led to the development of PMWS. These observations may contribute to a better understanding of the pathogenesis of a PCV2-infection.

Animals↗

PubMed2006

Yeast systems biology to unravel the network of life.

Systems biology focuses on obtaining a quantitative description of complete biological systems, even complete cellular function. In this way, it will be possible to perform computer-guided design of novel drugs, advanced therapies for treatment of complex diseases, and to perform in silico design of advanced cell factories for production of fuels, chemicals, food ingredients and pharmaceuticals. The yeast Saccharomyces cerevisiae represents an excellent model system; the density of biological information available on this organism allows it to serve as a eukaryotic model for studying human diseases. Furthermore, it serves as an industrial workhorse for production of a wide range of chemicals and pharmaceuticals. Systems biology involves the combination of novel experimental techniques from different disciplines as well as functional genomics, bioinformatics and mathematical modelling, and hence no single laboratory has access to all the necessary competences. For this reason the Yeast Systems Biology Network (YSBN) has been established. YSBN will coordinate research efforts in yeast systems biology and, through the recently obtained EU funding for a Coordination Action, it will be possible to set appropriate guidelines, establish an appropriate infrastructure for the network and organize courses, meetings and conferences that will consolidate the network and promote systems biology. This paper discusses the impacts of systems biology and how YSBN may play a role in the future development of the field.

European Union↗

PubMed2006

Cytokine and C-reactive protein profiles induced by porcine circovirus type 2 experimental infection in 3-week-old piglets.

The purpose of this study was to determine serum profiles of cytokines at a protein level and Creactive protein (CRP) during the development of postweaning multisystemic wasting syndrome (PMWS) in experimentally inoculated pigs. Levels of serum IFN-alpha, IL-6, IL-10, and CRP were examined for a 35-day period in 10 piglets experimentally infected with PCV2 at 3 weeks of age. Four of the infected piglets developed severe PMWS at 14 to 21 days post-infection (d.p.i.) and died prior to termination of the experiment. The remaining six PCV2-infected piglets experienced transient fever, but did not display overt clinical signs of PMWS and were considered as subclinically infected. A bioassay was used to detect IL-6 and ELISAs were used to detect IFN-alpha, IL-10, and CRP. There were no significant differences in cytokine or CRP expression from 0 to 7 d.p.i. between the PMWS-affected and the subclinically infected piglets. Levels of IL-10 and CRP were elevated from 10 and 14 d.p.i. respectively in the PMWS-affected piglets compared to the subclinically infected piglets. There were no significant differences in IFN-alpha and IL-6 expression between the PMWS-affected piglets and the subclinically infected piglets. The present study shows that elevated levels of serum CRP and IL-10 were associated with PCV2-infected piglets that subsequently developed severe PMWS. This may help to provide further insight into the immunoaetiogenesis of this syndrome.

Animals↗

PubMed2006

Sex differential effects of routine immunizations and childhood survival in rural Malawi.

BACKGROUND: Recent studies from West Africa suggest that routine vaccinations may have sex differential nontargeted effects. To answer whether these patterns are found elsewhere, we reanalyzed data about vaccinations and mortality in a Malawian cohort. METHODS: Children were followed from birth with monthly home visits until 18 months of age. Anthropometric measurements and vaccination information were obtained at these visits. We investigated survival in relation to vaccination status using 2 different methods. First, we included all children updating vaccination information retrospectively. Second, we included only those present at monthly home visits for whom prospective vaccination information was likely to be most complete. SUBJECTS: This study included 751 children surviving the first week of life. MAIN OUTCOME MEASURES: Main outcome measures consisted of mortality rate ratios (MR) for BCG, diphtheria-tetanus-pertussis (DTP) and measles-vaccinated children compared with children who had not received these vaccines and female-male MRs among children who had received the same vaccine. RESULTS: In all analyses, BCG and MV were associated with lower mortality. Children with better nutritional status received DTP1 significantly earlier (P = 0.02). However, DTP1-vaccinated children did not have lower mortality, the MR being 0.99 (0.45-2.19) and 3.19 (0.80-12.8), respectively, using the 2 analyses with retrospective updating or with prospective determination of vaccination status. The female-male MR varied significantly with age, being increased at 3 to 8 months of age, the age of DTP vaccination, and reduced between 9 and 17 months of age, the age of measles vaccination (MV). Using prospective vaccination information, the MR for girls declined after BCG vaccination of unvaccinated girls but increased after DTP1 (P = 0.10) and declined after MV (P = 0.01). Children who received DTP and MV simultaneously had a MR of 5.27 (1.11-25.0) compared with children who received MV only. CONCLUSION: The female-male mortality ratio varied with age. The mode of analysis was important. Using prospective vaccination information like in previous studies from West Africa, BCG, DTP1 and MV had differential effects for girls and boys.

Diphtheria-Tetanus-Pertussis Vaccine↗

PubMed2006

Long-term treatment of pigs with low doses of monoclonal antibodies against porcine CD4 and CD8 antigens.

In vivo depletion of lymphocyte subsets allows investigation of the role of specific subsets in protective immunity. In the present study we evaluated the effects of long-term, low-dose treatment with murine monoclonal antibodies (mAbs) against porcine CD4 and CD8 surface antigens on lymphocyte subsets in pigs. Four-week-old pigs were treated by intramuscular injections of hybridoma cell culture supernatants containing anti-CD mAbs twice a week for a period of 5 weeks. The immunomodulatory effects of the treatments were assessed by flow cytometry (FCM) analysis of peripheral blood lymphocytes. Treatment with the anti-CD4 mAb almost completely eliminated the CD4(+) T-cell subset from the circulation after 2 weeks of therapy. This depletion persisted until the end of the experimental period 5 weeks after initiated therapy. Treatment with the anti-CD8 mAb was less effective, reducing the CD8(+) T-cell subset in peripheral blood by approximately 50% of the initial level after 3 weeks of therapy. Further, the anti-CD8 mAb-treated pigs showed a parallel increase in the CD4(+) T-cell subset from day 7. Two-colour FCM analysis indicated that a shift in phenotype from single-positive CD4(+)/CD8(-) to double-positive CD4(+)/CD8(+) T-cells might have occurred in these pigs. In the present experiment we demonstrated specific modulation of the peripheral blood T-lymphocyte population in pigs with continuous low-dose injections of specific mAb. The ability to modulate individual T-cell subsets should provide a method to elucidate their functionality in protection against infectious disease.

Animals↗

PubMed2006

Global transcriptional and physiological responses of Saccharomyces cerevisiae to ammonium, L-alanine, or L-glutamine limitation.

The yeast Saccharomyces cerevisiae encounters a range of nitrogen sources at various concentrations in its environment. The impact of these two parameters on transcription and metabolism was studied by growing S. cerevisiae in chemostat cultures with l-glutamine, l-alanine, or l-ammonium in limitation and by growing cells in an excess of ammonium. Cells grown in l-alanine-limited cultures had higher biomass yield per nitrogen mole (19%) than those from ammonium-limited cultures. Whole-genome transcript profiles were analyzed with a genome-scale metabolic model that suggested increased anabolic activity in l-alanine-limited cells. The changes in these cells were found to be focused around pyruvate, acetyl coenzyme A, glyoxylate, and alpha-ketoglutarate via increased levels of ALT1, DAL7, PYC1, GDH2, and ADH5 and decreased levels of GDH3, CIT2, and ACS1 transcripts. The transcript profiles were then clustered. Approximately 1,400 transcripts showed altered levels when amino acid-grown cells were compared to those from ammonium. Another 400 genes had low transcript levels when ammonium was in excess. Overrepresentation of the GATAAG element in their promoters suggests that nitrogen catabolite repression (NCR) may be responsible for this regulation. Ninety-one genes had transcript levels on both l-glutamine and ammonium that were decreased compared to those on l-alanine, independent of the concentration. The GATAAG element in these genes suggests two groups of NCR-responsive genes, those that respond to high levels of nitrogen and those that respond to levels below 30 muM. In conclusion, our results reveal that the nitrogen source has substantial influence on the transcriptome of yeasts and that transcriptional changes may be correlated to physiology via a metabolic model.

Alanine↗

PubMed2006

Deletion of RTS1, encoding a regulatory subunit of protein phosphatase 2A, results in constitutive amino acid signaling via increased Stp1p processing.

In Saccharomyces cerevisiae, extracellular amino acids are sensed at the plasma membrane by the SPS sensor, consisting of the transporter homologue Ssy1p, Ptr3p, and the endoprotease Ssy5p. Amino acid sensing results in proteolytic truncation of the transcription factors Stp1p and Stp2p, followed by their relocation from the cytoplasm to the nucleus, where they activate transcription of amino acid permease genes. We screened a transposon mutant library for constitutively signaling mutants, with the aim of identifying down-regulating components of the SPS-mediated pathway. Three isolated mutants were carrying a transposon in the RTS1 gene, which encodes a regulatory subunit of protein phosphatase 2A. We investigated the basal activity of the AGP1 and BAP2 promoters in rts1delta cells and found increased transcription from these promoters, as well as increased Stp1p processing, even in the absence of amino acids. Based on our findings we propose that the phosphatase complex containing Rts1p keeps the SPS-mediated pathway down-regulated in the absence of extracellular amino acids by dephosphorylating a component of the pathway.

Amino Acid Transport Systems↗

PubMed2005

Evolutionary programming as a platform for in silico metabolic engineering.

BACKGROUND: Through genetic engineering it is possible to introduce targeted genetic changes and hereby engineer the metabolism of microbial cells with the objective to obtain desirable phenotypes. However, owing to the complexity of metabolic networks, both in terms of structure and regulation, it is often difficult to predict the effects of genetic modifications on the resulting phenotype. Recently genome-scale metabolic models have been compiled for several different microorganisms where structural and stoichiometric complexity is inherently accounted for. New algorithms are being developed by using genome-scale metabolic models that enable identification of gene knockout strategies for obtaining improved phenotypes. However, the problem of finding optimal gene deletion strategy is combinatorial and consequently the computational time increases exponentially with the size of the problem, and it is therefore interesting to develop new faster algorithms. RESULTS: In this study we report an evolutionary programming based method to rapidly identify gene deletion strategies for optimization of a desired phenotypic objective function. We illustrate the proposed method for two important design parameters in industrial fermentations, one linear and other non-linear, by using a genome-scale model of the yeast Saccharomyces cerevisiae. Potential metabolic engineering targets for improved production of succinic acid, glycerol and vanillin are identified and underlying flux changes for the predicted mutants are discussed. CONCLUSION: We show that evolutionary programming enables solving large gene knockout problems in relatively short computational time. The proposed algorithm also allows the optimization of non-linear objective functions or incorporation of non-linear constraints and additionally provides a family of close to optimal solutions. The identified metabolic engineering strategies suggest that non-intuitive genetic modifications span several different pathways and may be necessary for solving challenging metabolic engineering problems.

Algorithms↗

PubMed2005

Mortality patterns during a war in Guinea-Bissau 1998-99: changes in risk factors?

BACKGROUND: The crude mortality rate of the whole population and the mortality of children <5 years of age are the common indicators of the severity of a complex emergency situation. However, these indicators rarely take account of differences in socioeconomic conditions and vulnerability. METHODS: We followed a population in Guinea-Bissau, which fled when fighting took place in the capital during the war in 1998-99. The population stayed close to the area of conflict and returned as soon as a cease-fire was negotiated. A peace treaty was signed after half-a-year. The following 6 months was a period of returning and re-settlement, even though two outbreaks of fighting occurred. RESULTS: In the first half-year the mortality rate was 78% [mortality ratio (MR) = 1.78; 95% CI 1.61-1.97] increased and mortality for children <5 years of age doubled (MR = 2.07; 95% CI 1.79-2.38). In the last 6 months of the war, mortality was slightly increased for children and not at all for the total population. In the first half-year, households living in better houses and having members with schooling were less affected. In the 're-settlement' period two inequalities emerged; the largest ethnic group, Pepel, continued to have high mortality when the mortality of other groups declined; likewise girls continued to have an elevated mortality whereas mortality of boys declined. CONCLUSION: Whereas specific 'free' interventions reduced social inequalities for the groups affected, for the total population health-inequalities were slightly amplified during the war. Once the population returned to their urban homes, mortality fell to pre-war levels even though some fighting continued, limited humanitarian aid was available and the pre-war infra-structure had not been re-established.

Child, Preschool↗

PubMed2005

In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production.

In silico genome-scale cell models are promising tools for accelerating the design of cells with improved and desired properties. We demonstrated this by using a genome-scale reconstructed metabolic network of Saccharomyces cerevisiae to score a number of strategies for metabolic engineering of the redox metabolism that will lead to decreased glycerol and increased ethanol yields on glucose under anaerobic conditions. The best-scored strategies were predicted to completely eliminate formation of glycerol and increase ethanol yield with 10%. We successfully pursued one of the best strategies by expressing a non-phosphorylating, NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase in S. cerevisiae. The resulting strain had a 40% lower glycerol yield on glucose while the ethanol yield increased with 3% without affecting the maximum specific growth rate. Similarly, expression of GAPN in a strain harbouring xylose reductase and xylitol dehydrogenase led to an improvement in ethanol yield by up to 25% on xylose/glucose mixtures.

Computer Simulation↗

PubMed2005

Global metabolite analysis of yeast: evaluation of sample preparation methods.

Sample preparation is considered one of the limiting steps in microbial metabolome analysis. Eukaryotes and prokaryotes behave very differently during the several steps of classical sample preparation methods for analysis of metabolites. Even within the eukaryote kingdom there is a vast diversity of cell structures that make it imprudent to blindly adopt protocols that were designed for a specific group of microorganisms. We have therefore reviewed and evaluated the whole sample preparation procedures for analysis of yeast metabolites. Our focus has been on the current needs in metabolome analysis, which is the analysis of a large number of metabolites with very diverse chemical and physical properties. This work reports the leakage of intracellular metabolites observed during quenching yeast cells with cold methanol solution, the efficacy of six different methods for the extraction of intracellular metabolites, and the losses noticed during sample concentration by lyophilization and solvent evaporation. A more reliable procedure is suggested for quenching yeast cells with cold methanol solution, followed by extraction of intracellular metabolites by pure methanol. The method can be combined with reduced pressure solvent evaporation and therefore represents an attractive sample preparation procedure for high-throughput metabolome analysis of yeasts.

Freeze Drying↗