1. |
- Marques, Wesley Leoricy, et al.
(author)
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Laboratory evolution and physiological analysis of Saccharomyces cerevisiae strains dependent on sucrose uptake via the Phaseolus vulgaris Suf1 transporter
- 2018
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In: Yeast. - : Wiley. - 0749-503X .- 1097-0061. ; 35:12, s. 639-652
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Journal article (peer-reviewed)abstract
- Knowledge on the genetic factors important for the efficient expression of plant transporters in yeast is still very limited. Phaseolus vulgaris sucrose facilitator 1 (PvSuf1), a presumable uniporter, was an essential component in a previously published strategy aimed at increasing ATP yield in Saccharomyces cerevisiae. However, attempts to construct yeast strains in which sucrose metabolism was dependent on PvSUF1 led to slow sucrose uptake. Here, PvSUF1-dependent S. cerevisiae strains were evolved for faster growth. Of five independently evolved strains, two showed an approximately twofold higher anaerobic growth rate on sucrose than the parental strain (mu = 0.19 h(-1) and mu = 0.08 h(-1), respectively). All five mutants displayed sucrose-induced proton uptake (13-50 mu mol H+ (g biomass)(-1) min(-1)). Their ATP yield from sucrose dissimilation, as estimated from biomass yields in anaerobic chemostat cultures, was the same as that of a congenic strain expressing the native sucrose symporter Mal11p. Four out of six observed amino acid substitutions encoded by evolved PvSUF1 alleles removed or introduced a cysteine residue and may be involved in transporter folding and/or oligomerization. Expression of one of the evolved PvSUF1 alleles (PvSUF1(I209F C265F G326C)) in an unevolved strain enabled it to grow on sucrose at the same rate (0.19 h(-1)) as the corresponding evolved strain. This study shows how laboratory evolution may improve sucrose uptake in yeast via heterologous plant transporters, highlights the importance of cysteine residues for their efficient expression, and warrants reinvestigation of PvSuf1's transport mechanism.
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2. |
- Bender, Nicole, et al.
(author)
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Chlamydia infection, pelvic inflammatory disease, ectopic pregnancy and infertility : cross-national study
- 2011
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In: Sexually Transmitted Infections. - : BMJ. - 1368-4973 .- 1472-3263. ; 87:7, s. 601-608
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Journal article (peer-reviewed)abstract
- Objectives To describe, using routine data in selected countries, chlamydia control activities and rates of chlamydia infection, pelvic inflammatory disease (PID), ectopic pregnancy and infertility and to compare trends in chlamydia positivity with rates of PID and ectopic pregnancy. Methods Cross-national comparison including national data from Australia, Denmark, the Netherlands, New Zealand, Sweden and Switzerland. Routine data sources about chlamydia diagnosis and testing and International Classification of Disease-10 coded diagnoses of PID, ectopic pregnancy and infertility in women aged 15-39 years from 1999 to 2008 were described. Trends over time and relevant associations were examined using Poisson regression. Results Opportunistic chlamydia testing was recommended in all countries except Switzerland, but target groups differed. Rates of chlamydia testing were highest in New Zealand. Chlamydia positivity was similar in all countries with available data (Denmark, New Zealand and Sweden) and increased over time. Increasing chlamydia positivity rates were associated with decreasing PID rates in Denmark and Sweden and with decreasing ectopic pregnancy rates in Denmark, New Zealand and Sweden. Ectopic pregnancy rates appeared to increase over time in 15-19-year-olds in several countries. Trends in infertility diagnoses were very variable. Conclusions The intensity of recommendations about chlamydia control varied between countries but was not consistently related to levels of chlamydia diagnosis or testing. Relationships between levels of chlamydia infection and complication rates between or within countries over time were not straightforward. Development and validation of indicators of chlamydia-related morbidity that can be compared across countries and over time should be pursued.
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3. |
- Marques, Wesley Leoricy, et al.
(author)
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Elimination of sucrose transport and hydrolysis in Saccharomyces cerevisiae : a platform strain for engineering sucrose metabolism
- 2017
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In: FEMS yeast research (Print). - : Oxford University Press. - 1567-1356 .- 1567-1364. ; 17:1
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Journal article (peer-reviewed)abstract
- Many relevant options to improve efficacy and kinetics of sucrose metabolism in Saccharomyces cerevisiae and, thereby, the economics of sucrose-based processes remain to be investigated. An essential first step is to identify all native sucrose-hydrolysing enzymes and sucrose transporters in this yeast, including those that can be activated by suppressor mutations in sucrose-negative strains. A strain in which all known sucrose-transporter genes (MAL11, MAL21, MAL31, MPH2, MPH3) were deleted did not grow on sucrose after 2 months of incubation. In contrast, a strain with deletions in genes encoding sucrose-hydrolysing enzymes (SUC2, MAL12, MAL22, MAL32) still grew on sucrose. Its specific growth rate increased from 0.08 to 0.25 h(-1) after sequential batch cultivation. This increase was accompanied by a 3-fold increase of in vitro sucrose-hydrolysis and isomaltase activities, as well as by a 3- to 5-fold upregulation of the isomaltase-encoding genes IMA1 and IMA5. One-step Cas9-mediated deletion of all isomaltase-encoding genes (IMA1-5) completely abolished sucrose hydrolysis. Even after 2 months of incubation, the resulting strain did not grow on sucrose. This sucrose-negative strain can be used as a platform to test metabolic engineering strategies and for fundamental studies into sucrose hydrolysis or transport.
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4. |
- Verhoeven, Maarten D., et al.
(author)
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Mutations in PMR1 stimulate xylose isomerase activity and anaerobic growth on xylose of engineered Saccharomyces cerevisiae by influencing manganese homeostasis
- 2017
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In: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Journal article (peer-reviewed)abstract
- Combined overexpression of xylulokinase, pentose-phosphate-pathway enzymes and a heterologous xylose isomerase (XI) is required but insufficient for anaerobic growth of Saccharomyces cerevisiae on d-xylose. Single-step Cas9-assisted implementation of these modifications yielded a yeast strain expressing Piromyces XI that showed fast aerobic growth on d-xylose. However, anaerobic growth required a 12-day adaptation period. Xylose-adapted cultures carried mutations in PMR1, encoding a Golgi Ca2+/Mn2+ ATPase. Deleting PMR1 in the parental XI-expressing strain enabled instantaneous anaerobic growth on d-xylose. In pmr1 strains, intracellular Mn2+ concentrations were much higher than in the parental strain. XI activity assays in cell extracts and reconstitution experiments with purified XI apoenzyme showed superior enzyme kinetics with Mn2+ relative to other divalent metal ions. This study indicates engineering of metal homeostasis as a relevant approach for optimization of metabolic pathways involving metal-dependent enzymes. Specifically, it identifies metal interactions of heterologous XIs as an underexplored aspect of engineering xylose metabolism in yeast.
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5. |
- Weeke, Lauren C., et al.
(author)
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Lidocaine response rate in aEEG-confirmed neonatal seizures : Retrospective study of 413 full-term and preterm infants
- 2016
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In: Epilepsia. - : Wiley. - 0013-9580 .- 1528-1167. ; 57:2, s. 233-242
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Journal article (peer-reviewed)abstract
- ObjectiveTo investigate the seizure response rate to lidocaine in a large cohort of infants who received lidocaine as second- or third-line antiepileptic drug (AED) for neonatal seizures. MethodsFull-term (n = 319) and preterm (n = 94) infants, who received lidocaine for neonatal seizures confirmed on amplitude-integrated EEG (aEEG), were studied retrospectively (January 1992-December 2012). Based on aEEG findings, the response was defined as good (>4 h no seizures, no need for rescue medication); intermediate (0-2 h no seizures, but rescue medication needed after 2-4 h); or no clear response (rescue medication needed <2 h). ResultsLidocaine had a good or intermediate effect in 71.4%. The response rate was significantly lower in preterm (55.3%) than in full-term infants (76.1%, p < 0.001). In full-term infants the response to lidocaine was significantly better than midazolam as second-line AED (21.4% vs. 12.7%, p = 0.049), and there was a trend for a higher response rate as third-line AED (67.6% vs. 57%, p = 0.086). Both lidocaine and midazolam had a higher response rate as third-line AED than as second-line AED (p < 0.001). Factors associated with a good response to lidocaine were the following: higher gestational age, longer time between start of first seizure and administration of lidocaine, lidocaine as third-line AED, use of new lidocaine regimens, diagnosis of stroke, use of digital aEEG, and hypothermia. Multivariable analysis of seizure response to lidocaine included lidocaine as second- or third-line AED and seizure etiology. SignificanceSeizure response to lidocaine was seen in similar to 70%. The response rate was influenced by gestational age, underlying etiology, and timing of administration. Lidocaine had a significantly higher response rate than midazolam as second-line AED, and there was a trend for a higher response rate as third-line AED. Both lidocaine and midazolam had a higher response rate as third-line compared to second-line AED, which could be due to a pharmacologic synergistic mechanism between the two drugs.
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