Protein phosphorylation is one of the most important post-translational modifications in nature. However, the site-specific incorporation of O-phosphotyrosine into proteins in vivo has not yet been reported. Endogenous phosphatases present in cells can dephosphorylate phosphotyrosine as a free amino acid or as a protein residue. Therefore, we deleted the genes of five phosphatases from the genome of Escherichia coli with the aim of stabilizing phosphotyrosine. Together with an engineered aminoacyl-tRNA synthetase (derived from Methanocaldococcus jannaschii tyrosyl-tRNA synthetase) and an elongation factor Tu variant, we were able to co-translationally incorporate O-phosphotyrosine into the super-folder green fluorescent protein at a desired position in vivo. This system will facilitate future studies of tyrosine phosphorylation.
These results demonstrate that rat gliomas can oxidize ketone bodies and indicate upregulation of ketone body transport when fed a ketogenic diet. Our findings contradict the hypothesis that brain tumors are metabolically inflexible and show the need for additional research on the use of ketogenic diets as therapy targeting brain tumor metabolism.
Incorporation of selenocysteine (Sec) in bacteria requires a UGA codon that is reassigned to Sec by the Sec-specific elongation factor SelB and a conserved mRNA motif (SECIS element). These requirements severely restrict the engineering of selenoproteins. Earlier a synthetic tRNASec was reported that allowed canonical Sec incorporation by EF-Tu; however, serine misincorporation limited its scope. We report a superior tRNASec variant (tRNAUTuX) that facilitates EF-Tu dependent stoichiometric Sec insertion in response to UAG both in vivo in Escherichia coli and in vitro in a cellfree protein synthesis system. We also demonstrate recoding of several sense codons in a SelB supplemented cell-free system. These advances in Sec incorporation will aid rational design and directed evolution of selenoproteins.
Background: Cardiovascular complications account for a significant proportion of the shortened lifespan of Marfan syndrome (MFS) patients, with aortic dissection being the most dreadful complication. The aortic root dilates initially in MFS patients, and given its important hemodynamic role, this can lead to aortic regurgitation and poses a substantial risk of aortic dissection. This study seeks to evaluate the natural history of aortic root aneurysms in MFS patients, with a focus on growth rates and correlation of root diameter with the risk of developing aortic complications.Methods: Seventy-eight patients confirmed to have MFS and aortic root dilatation were retrospectively reviewed, and their aortic root diameters serially analyzed. Annual growth rate estimates and yearly rates of adverse events were computed and correlated with aortic diameter. Results:The mean annual growth rate of the aortic root was estimated to be 0.26±0.05 cm/year (range 0.13 to 0.35 cm). Larger aneurysms grew faster, reaching up to 0.46 cm/year for aneurysms >6 cm. Mean age at onset of aortic dissection was 36±4 years. Annual rates of adverse events (rupture, dissection and death) were obtained using a logistic regression model at sizes 3.5, 4, 4.5, 5, 5.5 and 6 cm. A sharp increase of 23% in the probability of the risk of complications at diameters 5.5 to 6 cm was recognized.Conclusions: Aortic root aneurysms in MFS patients tend to have a faster expansion rate compared to non-MFS individuals, with aortic root diameter having a significant impact on the yearly risk of developing aortic complications.
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