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61.
High-throughput generation of sequence indexes from T-DNA mutagenized Arabidopsis thaliana lines 总被引:3,自引:0,他引:3
A pipeline has been created for the characterization of Arabidopsis thaliana mutants by generating flanking sequence tags (FSTs) and optimized for economic, high-throughput production. The GABI-Kat collection of T-DNA mutagenized A. thaliana plants was used as a source of independent transgenic lines. The pipeline included robotized extraction of genomic DNA in a 96-well format, an adapter-ligation PCR method for amplification of plant sequences adjacent to T-DNA borders, automated purification and sequencing of PCR products, and computational trimming of the resulting sequence files. Data quality was significantly improved by (i) restriction digestion of the adaptor-ligation products to reduce trivial sequences caused by co-amplification of fragments derived from the free plasmid, and (ii) the design of the adaptor primers for the second amplification step to enhance selective generation of single PCR fragments, even from lines with multiple T-DNA insertions. Gel-purification was avoided by including these steps, the number of amplification reactions per line was reduced from four to three, and the percentage of lines that yielded at least one FST was increased from 66% to 86%. More than 58,000 FSTs have been submitted to GenBank and are available at http://www.mpiz-koeln.mpg.de/GABI-Kat/. 相似文献
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Oxygen concentration profiles have been measured, by means of with microelectrodes in sediments of Lake Baikal and Lake Malawi,
along transects allowing to give a survey of two major ancient Rift lakes: Lake Baikal (Eastern Siberia) and Lake Malawi (East
Africa), along depth transects in the constitutive basins of the lakes and/or of relevant depths with regard to oxygen (including
including the deepest point, 1680 m, in Lake Baikal). Sediment oxygen penetration depths (SOPs) display very different patterns,
depending on the lake in the two lakes. In Lake Baikal, SOPs are variable, show no significant relationship with bathymetric
depth and are surprisingly deep on Akademichesky ridge (> 50.0 mm), emphasizing the distinctive feature of this region in
the lake. While the Selenga river is an important source of eutrophication, the similarity of SOP-values in the Selenga shallow
with those of most other sites suggests either a dilution of organic material by allochthonous matter, or a strong south-to-north
transport of particles. In Lake Malawi, available oxygen is restricted to a maximum of three millimetres of the sediment,
and there is a negative relationship with bathymetric depth, as a result of a steady decline of oxygen concentration with
depth through the water column. Amongst the few parameters known to affect SOPs, the oxygen consumption by the sediment seems
the most significant in both lakes. SOP-values furthermore confirm differences in the trophic status of Baikal and Malawi,
respectively. The importance of oxygen as a factor likely to create ecological segregation for benthic organisms is discussed.
Lake Malawi offers possibilities of bathymetric segregation but no vertical segregation in the sediment. In contrast, no bathymetric
segregation related to oxygen is possible in Lake Baikal, but vertical segregation in the sediment is very likely.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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66.
David Bednar Koen Beerens Eva Sebestova Jaroslav Bendl Sagar Khare Radka Chaloupkova Zbynek Prokop Jan Brezovsky David Baker Jiri Damborsky 《PLoS computational biology》2015,11(11)
There is great interest in increasing proteins’ stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt’s reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔT
m = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications. 相似文献
67.
Kim Beernaert Luc Deliens Koen Pardon Lieve Van den Block Dirk Devroey Kenneth Chambaere Joachim Cohen 《PloS one》2015,10(9)
Background
Many people who might benefit from specialist palliative care services are not using them.Aim
We examined the use of these services and the reasons for not using them in a population in potential need of palliative care.Methods
We conducted a population-based survey regarding end-of-life care among physicians certifying a large representative sample (n = 6188) of deaths in Flanders, Belgium.Results
Palliative care services were not used in 79% of cases of people with organ failure, 64% of dementia and 44% of cancer. The most frequently indicated reasons were that 1) existing care already sufficiently addressed palliative and supportive needs (56%), 2) palliative care was not deemed meaningful (26%) and 3) there was insufficient time to initiate palliative care (24%). The reasons differed according to patient characteristics: in people with dementia the consideration of palliative care as not meaningful was more likely to be a reason for not using it; in older people their care needs already being sufficiently addressed was more likely to be a reason. For those patients who were referred the timing of referral varied from a median of six days before death (organ failure) to 16 days (cancer).Conclusions
Specialist palliative care is not initiated in almost half of the people for whom it could be beneficial, most frequently because physicians deem regular caregivers to be sufficiently skilled in addressing palliative care needs. This would imply that the safeguarding of palliative care skills in this regular ‘general’ care is an essential health policy priority. 相似文献68.
69.
Jessica A. Hartmann Marieke Wichers Claudia Menne-Lothmann Ingrid Kramer Wolfgang Viechtbauer Frenk Peeters Koen R. J. Schruers Alex L. van Bemmel Inez Myin-Germeys Philippe Delespaul Jim van Os Claudia J. P. Simons 《PloS one》2015,10(6)
Objectives
Positive affect (PA) plays a crucial role in the development, course, and recovery of depression. Recently, we showed that a therapeutic application of the experience sampling method (ESM), consisting of feedback focusing on PA in daily life, was associated with a decrease in depressive symptoms. The present study investigated whether the experience of PA increased during the course of this intervention.Design
Multicentre parallel randomized controlled trial. An electronic random sequence generator was used to allocate treatments.Settings
University, two local mental health care institutions, one local hospital.Participants
102 pharmacologically treated outpatients with a DSM-IV diagnosis of major depressive disorder, randomized over three treatment arms.Intervention
Six weeks of ESM self-monitoring combined with weekly PA-focused feedback sessions (experimental group); six weeks of ESM self-monitoring combined with six weekly sessions without feedback (pseudo-experimental group); or treatment as usual (control group).Main outcome
The interaction between treatment allocation and time in predicting positive and negative affect (NA) was investigated in multilevel regression models.Results
102 patients were randomized (mean age 48.0, SD 10.2) of which 81 finished the entire study protocol. All 102 patients were included in the analyses. The experimental group did not show a significant larger increase in momentary PA during or shortly after the intervention compared to the pseudo-experimental or control groups (χ2 (2) =0.33, p=.846). The pseudo-experimental group showed a larger decrease in NA compared to the control group (χ2 (1) =6.29, p=.012).Conclusion
PA-focused feedback did not significantly impact daily life PA during or shortly after the intervention. As the previously reported reduction in depressive symptoms associated with the feedback unveiled itself only after weeks, it is conceivable that the effects on daily life PA also evolve slowly and therefore were not captured by the experience sampling procedure immediately after treatment.Trial Registration
Trialregister.nl/trialreg/index.asp. NTR1974 相似文献70.
Janneke A. Cox Robert L. Lukande Sam Kalungi Eric Van Marck Koen Van de Vijver Andrew Kambugu Ann M. Nelson Robert Colebunders Yukari C. Manabe 《PloS one》2015,10(4)