Extrinsic Fluorescent Dyes as Tools for Protein Characterization

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

S9.6-based hybrid capture immunoassay for pathogen detection

Abstract The detection of pathogens is critical for clinical diagnosis and public health surveillance. Detection is usually done with nucleic acid-based tests (NATs) and rapid antigen tests (e.g., lateral flow assays [LFAs]). Although NATs are more sensitive and specific, their use is often limited in resource-poor settings due to specialized requirements. To address this limitation, we developed a rapid DNA-RNA Hybrid Capture immunoassay (HC) that specifically detects RNA from pathogens. This assay utilizes a unique monoclonal antibody, S9.6, which binds DNA-RNA hybrids. Biotinylated single-stranded DNA probes are hybridized to target RNAs, followed by hybrid capture on streptavidin and detection with S9.6. The HC-ELISA assay can detect as few as 104 RNA molecules that are 2.2 kb in length. We also adapted this assay into a LFA format, where captured Bacillus anthracis rpoB RNA of 3.5 kb length was detectable from a bacterial load equivalent to 107 CFU per 100 mg of mouse tissue using either HC-ELISA or HC-LFA. Importantly, we also demonstrated the versatility of HC by detecting other pathogens, including SARS-CoV-2 and Toxoplasma gondii, showing its potential for broad pathogen detection. Notably, HC does not require amplification of the target nucleic acid and utilizes economical formats like ELISA and LFA, making it suitable for use in sentinel labs for pathogen detection or as a molecular tool in basic research laboratories. Our study highlights the potential of HC as a sensitive and versatile method for RNA-based pathogen detection

S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

Diagnosis of infectious agents is increasingly done by the detection of unique nucleic acid sequences, typically using methods such as PCR that specifically amplify these sequences. A largely neglected alternative approach is to use antibodies that recognize nucleic acids. The unique monoclonal antibody S9.6 recognizes DNA–RNA hybrids in a largely sequence-independent manner. S9.6 has been used in several cases for the analysis of nucleic acids. Extending our recent determination of the structure of S9.6 Fab bound to a DNA–RNA hybrid, we have developed reagents and methods for the sensitive detection of specific DNA and RNA sequences. To facilitate the use in diagnostics, we conjugated the S9.6 Fab to the highly active and well-characterized reporter enzyme human-secreted embryonic alkaline phosphatase (SEAP). Two approaches were utilized for conjugation. The first used sortase A (SrtA), which generates a covalent peptide bond between short amino acid sequences added to recombinantly produced S9.6 Fab and SEAP. The second approach was to genetically fuse the S9.6 Fab and SEAP so that the two are produced as a single molecule. Using these two antibody–SEAP proteins, we developed a simplified ELISA format for the identification of synthetic DNA–RNA hybrids, which can be optimized for detecting nucleic acids of pathogens, as well as for other applications. We successfully used this immunosorbent assay, HC-S, to identify DNA–RNA hybrids in solution with high specificity and sensitivity