8 research outputs found
Other Mechanical Methods for Pre-Induction Cervical Ripening.
Pre-induction cervical ripening is an important part of the labor induction process in women with an unfavorable cervix. This can be achieved either by pharmacologic or mechanical methods of cervical ripening. While the Foley catheter is the most commomly used mechanical method for labor induction, other mechanical methods are also available. This article reviews the safety profiles of osmotic dilators, extra-amniotic saline infusion, double-balloon catheters, and also compares their efficacy to that of other mechanical and pharmacologic cervical ripening methods. While mechanical methods have been shown to be safe and effective for cervical ripening, none of these alternatives has been shown to be superior to the Foley catheter
Directed evolution of a probe ligase with activity in the secretory pathway and application to imaging intercellular protein-protein interactions.
Previously, we reported a new method for intracellular protein labeling in living cells called PRIME (probe incorporation mediated by enzymes). PRIME uses a mutant of Escherichia coli lipoic acid ligase (LplA) to catalyze covalent probe ligation onto a 13-amino acid peptide recognition sequence. While our first demonstration labeled proteins with a coumarin fluorophore, subsequent engineering produced alkyl azide and trans-cyclooctene ligases as well as an interaction-dependent form of the coumarin PRIME method (ID-PRIME). One major limitation of the PRIME methodologies is that LplA mutants have very low activity in the secretory pathway. Here, we extend PRIME labeling to oxidizing compartments such as the endoplasmic reticulum and the cell surface. We used yeast-display evolution and four rounds of selection to isolate LplA mutants with improved picolyl azide ligation activity. Then we compared the ligation activities of the evolved mutants both in vitro and on the mammalian cell surface. We characterized the picolyl azide ligation activity of the most active LplA variant in vitro, in the endoplasmic reticulum, and at the mammalian cell surface. Finally, we used the optimized LplA variant to label neurexin and neuroligin interactions at the mammalian cell surface in just 5 min. Compared to another method for imaging these protein-protein interactions (GFP recomplementation across synapses), our optimized ID-PRIME ligase is faster, more sensitive, and does not trap interacting proteins in a complex (nontrapping)
Directed Evolution of a Probe Ligase with Activity in the Secretory Pathway and Application to Imaging Intercellular Protein–Protein Interactions
Previously,
we reported a new method for intracellular protein
labeling in living cells called PRIME (probe incorporation mediated
by enzymes). PRIME uses a mutant of <i>Escherichia coli</i> lipoic acid ligase (LplA) to catalyze covalent probe ligation onto
a 13-amino acid peptide recognition sequence. While our first demonstration
labeled proteins with a coumarin fluorophore, subsequent engineering
produced alkyl azide and <i>trans</i>-cyclooctene ligases
as well as an interaction-dependent form of the coumarin PRIME method
(ID-PRIME). One major limitation of the PRIME methodologies is that
LplA mutants have very low activity in the secretory pathway. Here,
we extend PRIME labeling to oxidizing compartments such as the endoplasmic
reticulum and the cell surface. We used yeast-display evolution and
four rounds of selection to isolate LplA mutants with improved picolyl
azide ligation activity. Then we compared the ligation activities
of the evolved mutants both <i>in vitro</i> and on the mammalian
cell surface. We characterized the picolyl azide ligation activity
of the most active LplA variant <i>in vitro</i>, in the
endoplasmic reticulum, and at the mammalian cell surface. Finally,
we used the optimized LplA variant to label neurexin and neuroligin
interactions at the mammalian cell surface in just 5 min. Compared
to another method for imaging these protein–protein interactions
(GFP recomplementation across synapses), our optimized ID-PRIME ligase
is faster, more sensitive, and does not trap interacting proteins
in a complex (nontrapping)