Developing Nursing Care Guidelines for Patients With COVID-19.

The COVID-19 pandemic has produced an abundance of new and evolving evidence related to providing care for this complex patient population. Keeping up with the rapid flow of published information can be challenging and time-consuming, even for those skilled at interpreting the literature. To help clinical nurses readily apply standardized, evidence-based recommendations in a rapidly changing healthcare environment, the Good Samaritan Medical Center Education Team created a nursing-specific guideline for care of patients with COVID-19.

Potato leafroll virus structural proteins manipulate overlapping, yet distinct protein interaction networks during infection.

Potato leafroll virus (PLRV) produces a readthrough protein (RTP) via translational readthrough of the coat protein amber stop codon. The RTP functions as a structural component of the virion and as a nonincorporated protein in concert with numerous insect and plant proteins to regulate virus movement/transmission and tissue tropism. Affinity purification coupled to quantitative MS was used to generate protein interaction networks for a PLRV mutant that is unable to produce the read through domain (RTD) and compared to the known wild-type PLRV protein interaction network. By quantifying differences in the protein interaction networks, we identified four distinct classes of PLRV-plant interactions: those plant and nonstructural viral proteins interacting with assembled coat protein (category I); plant proteins in complex with both coat protein and RTD (category II); plant proteins in complex with the RTD (category III); and plant proteins that had higher affinity for virions lacking the RTD (category IV). Proteins identified as interacting with the RTD are potential candidates for regulating viral processes that are mediated by the RTP such as phloem retention and systemic movement and can potentially be useful targets for the development of strategies to prevent infection and/or viral transmission of Luteoviridae species that infect important crop species.

Evidence for lysine acetylation in the coat protein of a polerovirus.

Virions of the RPV strain of Cereal yellow dwarf virus-RPV were purified from infected oat tissue and analysed by MS. Two conserved residues, K147 and K181, in the virus coat protein, were confidently identified to contain epsilon-N-acetyl groups. While no functional data are available for K147, K181 lies within an interfacial region critical for virion assembly and stability. The signature immonium ion at m/z 126.0919 demonstrated the presence of N-acetyllysine, and the sequence fragment ions enabled an unambiguous assignment of the epsilon-N-acetyl modification on K181. We hypothesize that selection favours acetylation of K181 in a fraction of coat protein monomers to stabilize the capsid by promoting intermonomer salt bridge formation.

E. coli sabotages the in vivo production of O-linked ß-N-acetylglucosamine-modified proteins.

The O-linked ß-N-acetylglucosamine (O-GlcNAc) post-translational modification is an important, regulatory modification of cytosolic and nuclear enzymes. To date, no 3-dimensional structures of O-GlcNAc-modified proteins exist due to difficulties in producing sufficient quantities with either in vitro or in vivo techniques. Recombinant co-expression of substrate protein and O-GlcNAc transferase in Escherichia coli was used to produce O-GlcNAc-modified domains of human cAMP responsive element-binding protein (CREB1) and Abelson tyrosine-kinase 2 (ABL2). Recombinant expression in E. coli is an advantageous approach, but only small quantities of insoluble O-GlcNAc-modified protein were produced. Adding ß-N-acetylglucosaminidase inhibitor, O-(2-acetamido-2-dexoy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc), to the culture media provided the first evidence that an E. coli enzyme cleaves O-GlcNAc from proteins in vivo. With the inhibitor present, the yields of O-GlcNAc-modified protein increased. The E. coli ß-N-acetylglucosaminidase was isolated and shown to cleave O-GlcNAc from a synthetic O-GlcNAc-peptide in vitro. The identity of the interfering ß-N-acetylglucosaminidase was confirmed by testing a nagZ knockout strain. In E. coli, NagZ natively cleaves the GlcNAc-ß1,4-N-acetylmuramic acid linkage to recycle peptidoglycan in the cytoplasm and cleaves the GlcNAc-ß-O-linkage of foreign O-GlcNAc-modified proteins in vivo, sabotaging the recombinant co-expression system.

Methods to identify the NMR resonances of the ¹³C-dimethyl N-terminal amine on reductively methylated proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive (13)C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive (13)C-methylation is a chemical reaction performed under mild conditions that modifies a protein's primary amino groups (lysine ε-amino groups and the N-terminal α-amino group) to (13)C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive (13)C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein (13)C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of (13)C-labeling at each primary amino group. One limitation of the method arises when the protein's N-terminal residue is a lysine because the α- and ε-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the (13)C-dimethylamines associated with both the N-terminal α-amine and the side chain ε-amine. The NMR signals of the N-terminal α-dimethylamine and the side chain ε-dimethylamine of hen egg white lysozyme, Lys1, are identified in (1)H-(13)C heteronuclear single-quantum coherence spectra.

The recognition of nucleotides with model beta-hairpin receptors: investigation of critical contacts and nucleotide selectivity.

[reaction: see text] We have investigated the factors that contribute to binding of ATP by a designed 12-residue beta-hairpin peptide, WKWK, and have determined its selectivity for binding to the naturally occurring nucleotide triphosphates. We have previously shown that WKWK creates an ATP binding pocket on one face of the beta-hairpin consisting of two Trp and two Lys residues. Mutation of the two Lys residues on the binding face of the beta-hairpin resulted in a lower affinity, indicating that each is involved in ATP binding and that each residue contributes approximately -1.5 kcal/mol to the energy of complexation. Replacement of either Trp residue of the ATP binding pocket with Phe or Leu destabilizes the complex formed with ATP by approximately 1 kcal/mol, indicating that both Trp residues participate in interactions with ATP. For binding to the nucleotide triphosphates, the order of binding affinity was shown to follow dTTP > GTP > ATP > CTP, with differences in binding energies spanning as much as 1.6 kcal/mol. NMR analysis demonstrates that both aromatic interactions with the Trp side chains and CH-pi interactions between the ribose protons and the Trp residues may contribute significantly to binding. The results from our model system provide useful thermodynamic information regarding protein-nucleic acid interactions that occur at the surface of a beta-sheet.