| 181. |
- Smith, Cory J., et al.
(författare)
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Enabling large-scale genome editing at repetitive elements by reducing DNA nicking
- 2020
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:9, s. 5183-5195
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Tidskriftsartikel (refereegranskat)abstract
- To extend the frontier of genome editing and enable editing of repetitive elements of mammalian genomes, we made use of a set of dead-Cas9 base editor (dBE) variants that allow editing at tens of thousands of loci per cell by overcoming the cell death associated with DNA double-strand breaks and single-strand breaks. We used a set of gRNAs targeting repetitive elements-ranging in target copy number from about 32 to 161 000 per cell. dBEs enabled survival after large-scale base editing, allowing targeted mutations at up to ∼13 200 and ∼12 200 loci in 293T and human induced pluripotent stem cells (hiPSCs), respectively, three orders of magnitude greater than previously recorded. These dBEs can overcome current on-target mutation and toxicity barriers that prevent cell survival after large-scale genome engineering.
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| 182. |
- Sollerman, Jesper, et al.
(författare)
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Observations of the Crab Nebula and Its Pulsar in the Far-Ultraviolet and in the Optical
- 2000
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Ingår i: Astrophysical Journal. - : American Astronomical Society. - 0004-637X .- 1538-4357. ; 537, s. 861-874
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Tidskriftsartikel (refereegranskat)abstract
- We present far-UV observations of the Crab Nebula and its pulsar made with the Space Telescope Imaging Spectrograph on board the Hubble Space Telescope. Broad, blueshifted absorption arising in the nebula is seen in C IV λ1550, reaching a blueward velocity of ~2500 km s-1. This can be interpreted as evidence for a fast outer shell surrounding the Crab Nebula, and we adopt a spherically symmetric model to constrain the properties of such a shell. From the line profile we find that the density appears to decrease outward in the shell. A likely lower limit to the shell mass is ~0.3 Msolar with an accompanying kinetic energy of ~1.5×1049 ergs. A fast massive shell with 1051 ergs cannot be excluded but is less likely if the density profile is much steeper than ρ(R)~R-4 and the maximum velocity is <~6000 km s-1. The observations cover the region 1140-1720 Å, which is further into the ultraviolet than has previously been obtained for the pulsar. With the time-tag mode of the spectrograph we obtain the pulse profile in this spectral regime. The profile is similar to that previously obtained by us in the near-UV, although the primary peak is marginally narrower. Together with the near-UV data, and new optical data from the Nordic Optical Telescope, our spectrum of the Crab pulsar covers the entire region from 1140 to 9250 Å. Dereddening the spectrum with a standard extinction curve we achieve a flat spectrum for the reddening parameters E(B-V)=0.52, R=3.1. This dereddened spectrum of the Crab pulsar can be fitted by a power law with spectral index αν=0.11+/-0.04. The main uncertainty in determining the spectral index is the amount and characteristics of the interstellar reddening, and we have investigated the dependence of αν on E(B-V) and R. In the extended emission covered by our 25''×0.5" slit in the far-UV, we detect C IV λ1550 and He II λ1640 emission lines from the Crab Nebula. Several interstellar absorption lines are detected along the line of sight to the pulsar. The Lyα absorption indicates a column density of (3.0+/-0.5)×1021 cm-2 of neutral hydrogen, which agrees well with our estimate of E(B-V)=0.52 mag. Other lines show no evidence of severe depletion of metals in atomic gas. Based on observations with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc. under NASA contract NAS5-26555.
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| 183. |
- Tavakoli, Meysam, et al.
(författare)
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Single Molecule Data Analysis : An Introduction
- 2017
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Ingår i: Advances in Chemical Physics. - Hoboken, USA : John Wiley & Sons. - 1934-4791. - 9781119324577 - 9781119324560 ; , s. 205-305
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Bokkapitel (refereegranskat)abstract
- This chapter considers statistical data-driven analysis methods, and focuses on parametric as well as more recent information theoretic and nonparametric statistical approaches to biophysical data analysis with an emphasis on single-molecule applications. It then reviews simpler parametric approaches starting from an assumed model with unknown parameters. Model selection criteria are widely used in biophysical data analysis from image deconvolution to single-molecule step detection and continue to be developed by statisticians. The goal of successful model selection criteria is to pick models whose complexity is penalized, in a principled fashion, to avoid overfitting and that convincingly fit the data provided (the training set). The chapter summarizes both information theoretic as well as Bayesian model selection criteria. Finally, the chapter discusses efforts to use information theory in experimental design and ends with some considerations on the broader applicability of information theory.
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| 184. |
- Thomas, Gemma L., et al.
(författare)
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Peptide ligations accelerated by N-terminal aspartate and glutamate residues.
- 2011
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Ingår i: Organic Letters. - : American Chemical Society (ACS). - 1523-7060 .- 1523-7052. ; 13:18, s. 4770-3
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Tidskriftsartikel (refereegranskat)abstract
- A novel application of intramolecular base catalysis confers enhanced reaction rates for aminolysis ligations between peptide thioesters and peptides bearing N-terminal aspartate or glutamate residues. The broad scope of this process and its application in the total synthesis of the diabetes drug exenatide is demonstrated.
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| 185. |
- Tomaz, Tiago, et al.
(författare)
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Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis.
- 2010
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Ingår i: Plant Physiology. - : American Society of Plant Biologists. - 0032-0889 .- 1532-2548. ; 154:3, s. 1143-1157
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Tidskriftsartikel (refereegranskat)abstract
- Malate dehydrogenase (MDH) catalyzes a reversible NAD(+)-dependent-dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments. To explore the role of mitochondrial MDH (mMDH) in Arabidopsis (Arabidopsis thaliana), knockout single and double mutants for the highly expressed mMDH1 and lower expressed mMDH2 isoforms were constructed and analyzed. A mmdh1mmdh2 mutant has no detectable mMDH activity but is viable, albeit small and slow growing. Quantitative proteome analysis of mitochondria shows changes in other mitochondrial NAD-linked dehydrogenases, indicating a reorganization of such enzymes in the mitochondrial matrix. The slow-growing mmdh1mmdh2 mutant has elevated leaf respiration rate in the dark and light, without loss of photosynthetic capacity, suggesting that mMDH normally uses NADH to reduce oxaloacetate to malate, which is then exported to the cytosol, rather than to drive mitochondrial respiration. Increased respiratory rate in leaves can account in part for the low net CO(2) assimilation and slow growth rate of mmdh1mmdh2. Loss of mMDH also affects photorespiration, as evidenced by a lower postillumination burst, alterations in CO(2) assimilation/intercellular CO(2) curves at low CO(2), and the light-dependent elevated concentration of photorespiratory metabolites. Complementation of mmdh1mmdh2 with an mMDH cDNA recovered mMDH activity, suppressed respiratory rate, ameliorated changes to photorespiration, and increased plant growth. A previously established inverse correlation between mMDH and ascorbate content in tomato (Solanum lycopersicum) has been consolidated in Arabidopsis and may potentially be linked to decreased galactonolactone dehydrogenase content in mitochondria in the mutant. Overall, a central yet complex role for mMDH emerges in the partitioning of carbon and energy in leaves, providing new directions for bioengineering of plant growth rate and a new insight into the molecular mechanisms linking respiration and photosynthesis in plants.
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| 186. |
- van der Wouden, Cathelijne H., et al.
(författare)
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Generating evidence for precision medicine : considerations made by the Ubiquitous Pharmacogenomics Consortium when designing and operationalizing the PREPARE study
- 2020
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Ingår i: Pharmacogenetics & Genomics. - 1744-6872 .- 1744-6880. ; 30:6, s. 131-144
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Tidskriftsartikel (refereegranskat)abstract
- Objectives Pharmacogenetic panel-based testing represents a new model for precision medicine. A sufficiently powered prospective study assessing the (cost-)effectiveness of a panel-based pharmacogenomics approach to guide pharmacotherapy is lacking. Therefore, the Ubiquitous Pharmacogenomics Consortium initiated the PREemptive Pharmacogenomic testing for prevention of Adverse drug Reactions (PREPARE) study. Here, we provide an overview of considerations made to mitigate multiple methodological challenges that emerged during the design.Methods An evaluation of considerations made when designing the PREPARE study across six domains: study aims and design, primary endpoint definition and collection of adverse drug events, inclusion and exclusion criteria, target population, pharmacogenomics intervention strategy, and statistical analyses.Results Challenges and respective solutions included: (1) defining and operationalizing a composite primary endpoint enabling measurement of the anticipated effect, by including only severe, causal, and drug genotype-associated adverse drug reactions; (2) avoiding overrepresentation of frequently prescribed drugs within the patient sample while maintaining external validity, by capping drugs of enrolment; (3) designing the pharmacogenomics intervention strategy to be applicable across ethnicities and healthcare settings; and (4) designing a statistical analysis plan to avoid dilution of effect by initially excluding patients without a gene–drug interaction in a gatekeeping analysis.Conclusion Our design considerations will enable quantification of the collective clinical utility of a panel of pharmacogenomics-markers within one trial as a proof-of-concept for pharmacogenomics-guided pharmacotherapy across multiple actionable gene–drug interactions. These considerations may prove useful to other investigators aiming to generate evidence for precision medicine.
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| 187. |
- Vazin, Tandis, et al.
(författare)
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Assessment of Stromal-Derived Inducing Activity in the Generation of Dopaminergic Neurons from Human Embryonic Stem Cells
- 2008
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 26:6, s. 1517-1525
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Tidskriftsartikel (refereegranskat)abstract
- Producing dopaminergic (DA) neurons is a major goal of human embryonic stem cell (hESC) research. DA neurons can be differentiated from hESC by coculture with the mouse PA6 stromal cell line; this differentiation-inducing effect is termed stromal-derived inducing activity (SDIA). The molecular and biochemical nature of SDIA is, however, unknown. Various studies have suggested that SDIA involves either a fixation-resistant component located on the PA6 cell surface or factors secreted into the medium by PA6 cells. To address this question, hESC were cocultured with PA6 cells for 12 days and then further differentiated with sonic hedgehog homolog, fibroblast growth factor-8, and glial cell line-derived neurotrophic factor. After 18 days, 34% of cells were tyrosine hydroxylase (TH)+. When PA6 cells were fixed or irradiated, the number of TH+ cells was decreased by threefold, whereas mitomycin-c treatment of feeder cells decreased the number of TH+ cells by 32%. The neural-inducing effect of PA6 cells, as monitored by β-III-tubulin expression, was minimally affected by mitomycin-c treatment or fixation but was decreased 50% by irradiation. Medium conditioned by PA6 cells was ineffective in differentiating TH+ cells when used alone. Conditioned medium combined with heparin and/or fixed PA6 cells produced TH+ cell differentiation, although less effectively than PA6 cell coculture. Thus, PA6 cell surface activity is required for neural differentiation of hESC, but secreted factors are required for the specific DA neuron-inducing effect.
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| 188. |
- Vazin, Tandis, et al.
(författare)
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Dopaminergic neurons derived from BG01V2 : a variant of human embryonic stem cell line BG01
- 2008
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Ingår i: Restorative Neurology and Neuroscience. - 0922-6028 .- 1878-3627. ; 26:6, s. 447-458
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Tidskriftsartikel (refereegranskat)abstract
- Background and Purpose: Human embryonic stem cells (hESC) areconsidered a renewable source of dopamine producing neurons, and are of particularinterest for their potential clinical use in Parkinson’s disease. In this study, wecharacterized human dopaminergic neurons generated by stromal-derived inducingactivity (SDIA) from BG01V2, a strain of human embryonic stem cell line, BG01,characterized by a chromosome 17 trisomy. Similar chromosomal changes have beenrepeatedly observed in hESC cultures in different laboratories, indicating the importanceof chromosome 17 for growth and adaptation of hESC to culture. Methods: Weinvestigated in vitro proliferation of differentiating cells using a BrDU incorporationassay, and monitored the cell population in long term cultures. Despite the cytogeneticabnormality, TH+ neurons were postmitotic at all stages of differentiation. After 30 daysof differentiation, cell division ceased in 91% of the overall population of cells in theculture, indicating intact cell cycle regulation. Results: Expression of midbrain specificmarker genes (Otx2, Pax5, Msx-1) showed differentiation of hESC-derived neuralprogenitor cells into midbrain specific dopamine neurons. These neurons expressed thedopamine transporter (DAT), and displayed functional DAT activity and electricalexcitability. Conclusions: TH+ cells derived from the BG01V2 hESC line using SDIAare postmitotic and have functional characteristics of normal dopaminergic neurons.
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| 189. |
- Wemple, Beverley C., et al.
(författare)
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Ecohydrological disturbances associated with roads : Current knowledge, research needs, and management concerns with reference to the tropics
- 2018
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Ingår i: Ecohydrology. - : Wiley. - 1936-0584 .- 1936-0592. ; 11:3
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Forskningsöversikt (refereegranskat)abstract
- Roads are a pervasive form of disturbance with potential to negatively affect ecohydrological processes. Some of the most rapid growth in road networks is occurring in developing countries, particularly in the tropics, where political agendas are often focused on strengthening the economy, improving infrastructure, bolstering national security, achieving self-sufficiency, and increasing citizen well-being, often at the expense of the environment. We review what is known about road impacts on ecohydrological processes, focusing on aquatic systems, both temperate and tropical. We present seven cases that represent the broader trends of road development and impacts in tropical settings. Many of these process dynamics and impacts are not different from those experienced in temperate settings, although the magnitude of impacts in the tropics may be amplified with intense rainfall and lack of best management practices applied to road construction/maintenance. Impacts of roads in tropical settings may also be unique because of particular organisms or ecosystems affected. We outline a set of best practices to improve road network management and provide recommendations for adopting an agenda of research and road management in tropical settings. Importantly, we call for incorporation of transdisciplinary approaches to further study the effects of roads on ecohydrological processes in the tropics. Specific emphasis should also be placed on collaboration with governments and developers that are championing road development to help identify the drivers of road expansion and thresholds of negative impact, as well as methods of sustainable road construction and maintenance.
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| 190. |
- Wollman, Adam J.M., et al.
(författare)
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Critical roles for EGFR and EGFR-HER2 clusters in EGF binding of SW620 human carcinoma cells
- 2022
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Ingår i: Journal of the Royal Society Interface. - 1742-5689 .- 1742-5662. ; 19, s. 34-74
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Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor (EGF) signalling regulates normal epithelial and other cell growth, with EGF receptor (EGFR) overexpression reported in many cancers. However, the role of EGFR clusters in cancer and their dependence on EGF binding is unclear. We present novel single-molecule total internal reflection fluorescence microscopy of (i) EGF and EGFR in living cancer cells, (ii) the action of anti-cancer drugs that separately target EGFR and human EGFR2 (HER2) on these cells and (iii) EGFR-HER2 interactions. We selected human epithelial SW620 carcinoma cells for their low level of native EGFR expression, for stable transfection with fluorescent protein labelled EGFR, and imaged these using single-molecule localization microscopy to quantify receptor architectures and dynamics upon EGF binding. Prior to EGF binding, we observe pre-formed EGFR clusters. Unexpectedly, clusters likely contain both EGFR and HER2, consistent with co-diffusion of EGFR and HER2 observed in a different model CHO-K1 cell line, whose stoichiometry increases following EGF binding. We observe a mean EGFR: EGF stoichiometry of approximately 4: 1 for plasma membrane-colocalized EGFR-EGF that we can explain using novel time-dependent kinetics modelling, indicating preferential ligand binding to monomers. Our results may inform future cancer drug developments.
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