ABSTRACT
Background: The world urgently requires surrogate markers to diagnose COVID-19 and predict its progression. The severity is not easily predicted via currently used biomarkers. Critical COVID-19 patients need to be screened for hyperinflammation to improve mortality but expensive cytokine measurement is not routinely conducted in most laboratories. The neutrophil-to-lymphocyte ratio (NLR) is a novel biomarker in patients with various diseases. We evaluated the diagnostic and prognostic accuracy of the NLR in COVID-19 patients.Methods: We searched for relevant articles in seven databases. The quantitative analysis was conducted if at least two studies were evaluating the NLR role in COVID-19.Results: We included 8,120 individuals, including 7,482 COVID-19 patients, from 32 articles. Patients with COVID-19 had significantly higher levels of NLR compared to negative individuals. Advanced COVID-19 stages had significantly higher levels of NLR than earlier stages.Expert Opinion: We found significantly higher levels of NLR in advanced stages compared to earlier stages of COVID-19 with good accuracy to diagnose and predict the disease outcome, especially mortality prediction. A close evaluation of critical SARS-CoV-2 patients and efficient early management are essential measures to decrease mortality. NLR could help in assessing the resource allocation in severe COVID-19 patients even in restricted settings.
Subject(s)
COVID-19/blood , COVID-19/mortality , Lymphocyte Count , Neutrophils , Adult , Aged , COVID-19/etiology , Female , Humans , Leukocyte Count , Male , Middle Aged , Prognosis , Sensitivity and Specificity , Severity of Illness IndexABSTRACT
The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.
Subject(s)
Escherichia coli/genetics , Glycation End Products, Advanced/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/isolation & purification , Amyloid/genetics , Amyloid/metabolism , Amyloidosis/metabolism , Cloning, Molecular , Escherichia coli/metabolism , Extracellular Space/chemistry , Extracellular Space/genetics , Extracellular Space/metabolism , Glycation End Products, Advanced/biosynthesis , Glycation End Products, Advanced/genetics , Humans , Immunoglobulin Variable Region/genetics , Ligands , Models, Chemical , Mutagenesis, Site-Directed , Peptide Fragments/biosynthesis , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Receptor for Advanced Glycation End Products , Receptors, Immunologic/biosynthesisABSTRACT
Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.
Subject(s)
Dynamins/chemistry , Proline/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Vesicular Transport/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , CSK Tyrosine-Protein Kinase , Cyclin-Dependent Kinase 5 , Cyclin-Dependent Kinases/chemistry , Cyclin-Dependent Kinases/metabolism , Dynamin I/chemistry , Dynamin II/chemistry , Endocytosis , GRB2 Adaptor Protein , Glutathione Transferase/metabolism , Kinetics , Models, Chemical , Molecular Sequence Data , Nerve Tissue Proteins/chemistry , Phosphorylation , Polymerase Chain Reaction , Protein Binding , Protein Isoforms , Protein Structure, Tertiary , Protein-Tyrosine Kinases/chemistry , Rats , Recombinant Proteins/chemistry , Sequence Homology, Amino Acid , Signal Transduction , Substrate Specificity , Surface Plasmon Resonance , Time Factors , src Homology Domains , src-Family KinasesABSTRACT
We have determined the first de novo position of the secondary quinone QB in the Rhodobacter sphaeroides reaction center (RC) using phases derived by the single wavelength anomalous dispersion method from crystals with selenomethionine substitution. We found that in frozen RC crystals, QB occupies primarily the proximal binding site. In contrast, our room temperature structure showed that QB is largely in the distal position. Both data sets were collected in dark-adapted conditions. We estimate that the occupancy of the QB site is 80% with a proximal: distal ratio of 4:1 in frozen RC crystals. We could not separate the effect of freezing from the effect of the cryoprotectants ethylene glycol or glycerol. These results could have far-reaching implications in structure/function studies of electron transfer in the acceptor quinone complex because the above are the most commonly used cryoprotectants in spectroscopic experiments.
