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Characterizing outcomes of grow older, sexual intercourse as well as psychosis signs and symptoms on thalamocortical practical on the web connectivity within youth.
Incidence of cerebral lesions was significantly higher in patients on NOAC compared with VKA (16% vs. 9.2%, respectively, p = 0.04), and in patients who had intraprocedural cardioversions compared with no cardivoersions (19.5% vs. 10.4%, respectively, p = 0.03). In multivariate analysis, both parameters were found to be independent risk factors for cerebral embolism. No significant difference between interrupted and uninterrupted NOAC administration could be detected. CONCLUSIONS In patients undergoing AF ablation, we identified the use of NOAC and intraprocedural cardioversion as independent risk factors for the occurrence of periprocedural cerebral embolic lesions.Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. selleckchem The development of various β-mercapto amino acid building blocks coupled with ligation-desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation-desulfurization sequence.Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.α-Synuclein is a small aggregation-prone protein associated with Parkinson's disease (PD). The protein's biochemical and biophysical properties can be heavily influenced by various types of posttranslational modification (PTMs) such as phosphorylation, ubiquitination, and glycosylation. To understand the site-specific effects of various PTMs have on the protein and its aggregation, obtaining a homogeneous sample of the protein of interest with the specific modification of interest is key. Expressed protein ligation (EPL) has emerged as robust tool for building synthetic proteins bearing site-specific modifications. Here, we outline our approach for building α-synuclein with site specific O-GlcNAc modifications, an intracellular subtype of glycosylation that has been linked to the inhibition of protein aggregation. More specifically, we provide specific protocols for the synthesis of α-synuclein bearing an O-GlcNAc modification at threonine 72, termed α-synuclein(gT72). However, this general approach utilizing two recombinant fragments and one synthetic peptide is applicable to other sites and types of modifications and should be transferable to various other protein targets, including aggregation prone proteins like tau and TDP-43.The posttranslational modification of cellular proteins by ubiquitin (Ub), called ubiquitylation, is indispensable for the normal growth and development of eukaryotic organisms. In order to conduct studies that elucidate the precise mechanistic roles for Ub, access to site-specifically and homogenously ubiquitylated proteins and peptides is critical. link2 However, the low abundance, heterogeneity, and dynamic nature of protein ubiquitylation are significant limitations toward such studies. Here we provide a facile expressed protein ligation method that does not require specialized apparatus and permits the rapid semisynthesis of ubiquitylated peptides by using the atom-efficient ligation auxiliary 2-aminooxyethanethiol.Nucleosomes, the basic unit of chromatin, contain a protein core of histone proteins, which are heavily posttranslationally modified. These modifications form a combinatorial language which defines the functional state of the underlying genome. As each histone type exists in two copies in a nucleosome, the modification patterns can differ between the individual histones, resulting in asymmetry and increasing combinatorial complexity. To systematically explore the regulation of chromatin regulatory enzymes (writers, erasers, or readers), chemically defined nucleosomes are required. We have developed strategies to chemically modify histones and control nucleosome assembly, thereby enabling the reconstitution of asymmetric histone modification patterns. Here, we report a detailed protocol for the modular assembly of such nucleosomes. Employing a three-segment ligation strategy for the semisynthesis of H3, coupled with the use of the protease cleavable "lnc-tag," we provide an efficient and traceless method for the controlled semisynthesis and reconstitution of asymmetrically modified nucleosomes.Classical approaches for probing protein phosphorylation events rely on phosphomimicking amino acids or enzymatic phosphorylation of proteins. In many cases, phosphomimicking amino acids inadequately imitate actual protein phosphorylation, whereas the latter method suffers from an inability to control site specificity and stoichiometry. To circumvent these shortcomings, chemical biological approaches have been developed to enable introduction of phosphorylated amino acids into proteins in a reliable and controlled way. Here, we describe methods to make semisynthetic, phosphorylated PDZ domains, covering expressed protein ligation (EPL) strategies involving modifications within the N-terminal or C-terminal regions. We also enclose protocols for the biophysical characterization of the semisynthetic phosphorylated PDZ domains to establish whether the introduced phosphorylation affects protein structure, stability, and function.Expressed protein ligation (EPL), using non-self-cleaving inteins, allows for the site-specific addition of customized chemical moieties to the termini of proteins. In this way, protein activity can be preserved while functionalizing the target protein with a wide range of chemical handles. Here, we describe methods for EPL-based modification of proteins produced by yeast, employing an engineered, non-self-cleaving intein known as 202-08. Methods for EPL modification of both yeast surface displayed and secreted proteins with bioorthogonal chemical groups are described. These methods allow for the site-specific modification of intein-fused proteins produced in yeast.The development of expressed protein ligation (EPL) widened the scope of questions that could be addressed by mechanistic biochemistry. Protein trans-splicing (PTS) relies on the same basic chemical principles, but utilizes split inteins to tracelessly ligate distinct peptide or polypeptide fragments together with native peptide bonds. Here we present a method to adapt PTS methodologies for their use in live cells, in order to deliver synthetic or native histone modifications. As an example, we provide a protocol to incorporate a small molecule fluorophore into chromatinized histones. The protocol should be easily adaptable to incorporate other modifications to chromatin in vivo.Protein semisynthesis is a powerful tool for studying proteins and has contributed to a better understanding of protein structure and function and also driven innovations in protein science. Expressed protein ligation (EPL) is a widely used method to generate chemically modified proteins. However, EPL has some limitations, particularly relevant to modify challenging proteins such as antibodies. The method termed streamlined expressed protein ligation (SEPL) overcomes some of the problems of EPL, and other methods of protein semisynthesis, to generate challenging modified proteins such as antibody-drug conjugates (ADCs). ADCs targeting highly cytotoxic molecules to cancer cells, offer an attractive strategy to selectively eliminate tumor cells with improved therapeutic index than the antibodies or cytotoxic molecules themselves. Despite the potential of ADCs, the development of such complex molecules is challenging. link3 We provide here protocols to prepare site-specifically modified ADCs by streamlined expressed protein ligation (SEPL), which does not require the incorporation of unnatural modifications into the antibody. Therefore, fully native antibodies, with only the desired cytotoxic molecules attached, can be generated.The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.Native chemical ligation (NCL) enables the direct chemical synthesis and semisynthesis of proteins of different sizes and compositions, streamlining the access to proteins containing posttranslational modifications (PTMs). NCL assembles peptide fragments through the chemoselective reaction of a C-terminal α-thioester peptide, prepared either by chemical synthesis or via intein-splicing technology, and a recombinant or synthetic peptide containing an N-terminal Cys. Whereas the generation of C-terminal α-thioester proteins can be achieved via the recombinant fusion of the sequence of interest to an intein domain, chemical methods can also be used for synthetically accessible proteins. The use of Fmoc solid-phase peptide synthesis (Fmoc-SPPS) to obtain α-thioester peptides requires the development of novel strategies to overcome the lability of the thioester bond toward piperidine Fmoc-removal conditions. These new synthetic methods enable the easy introduction of PTMs in the thioester fragment. In this chapter, we describe an approach for the synthesis and use of C-terminal α-N-acylbenzimidazolinone (Nbz) and α-N-acyl-N'-methylbenzimidazolinone (MeNbz) peptides in NCL.
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