Robust and High-Throughput Analytical Flow Proteomics Analysis of Cynomolgus Monkey and Human Matrices With Zeno SWATH Data-Independent Acquisition.

Modern mass spectrometers routinely allow deep proteome coverage in a single experiment. These methods are typically operated at nanoflow and microflow regimes, but they often lack throughput and chromatographic robustness, which is critical for large-scale studies. In this context, we have developed, optimized, and benchmarked LC-MS methods combining the robustness and throughput of analytical flow chromatography with the added sensitivity provided by the Zeno trap across a wide range of cynomolgus monkey and human matrices of interest for toxicological studies and clinical biomarker discovery. Sequential Window Acquisition of All Theoretical Fragment Ion Mass Spectra (SWATH) data-independent acquisition (DIA) experiments with Zeno trap activated (Zeno SWATH DIA) provided a clear advantage over conventional SWATH DIA in all sample types tested with improved sensitivity, quantitative robustness, and signal linearity as well as increased protein coverage by up to 9-fold. Using a 10-min gradient chromatography, up to 3300 proteins were identified in tissues at 2 µg peptide load. Importantly, the performance gains with Zeno SWATH translated into better biological pathway representation and improved the ability to identify dysregulated proteins and pathways associated with two metabolic diseases in human plasma. Finally, we demonstrate that this method is highly stable over time with the acquisition of reliable data over the injection of 1000+ samples (14.2 days of uninterrupted acquisition) without the need for human intervention or normalization. Altogether, Zeno SWATH DIA methodology allows fast, sensitive, and robust proteomic workflows using analytical flow and is amenable to large-scale studies.

Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.

High-sensitivity quantification of antisense oligonucleotides for pharmacokinetic characterization.

Aim: Antisense oligonucleotides (ASOs) are a fast-growing drug modality. Pharmacokinetic characterization and accurate quantification of ASOs is critical for drug development. LC-MS and hybridization immunoassays are common methods to quantify ASOs but may lack sensitivity. In this study we aimed to develop an ASO quantification method with improved sensitivity. Methods: We developed a branched DNA approach for ASO quantification and compared it with hybridization immunoassays. Results: The branched DNA assay showed significantly improved sensitivity, with LLOQ 31.25 pg/ml in plasma, 6.4-and 16-fold higher than dual-probe hybridization electrochemiluminescence and single-probe hybridization ELISA, respectively, with adequate precision, accuracy, selectivity and specificity and acceptable matrix interference. Conclusion: Branched DNA for ASO quantification has significantly higher sensitivity and lower hemolysis interference.

Disease can be caused by genetic mutations that lead to overproduction or underproduction of an aberrant protein. Antisense oligonucleotides (ASOs) are a relatively new class of drugs. While most current drugs act at the protein level, ASOs work at the RNA level and minimize synthesis of the aberrant protein. ASOs are small synthetic nucleotides that specifically bind and modify the target RNA. Quantification of ASOs is important in drug development to understand how much of the drug is in circulation or in the body after a certain period of time. While there are methods available to quantify ASOs, they lack sensitivity. We developed a method called 'branched DNA' to quantify ASOs, and compared it with known ASO quantification methods. We found that the branched DNA method showed improved sensitivity compared with other existing methods and is a reliable method to quantify ASOs. This method may be used in clinical trials when improved sensitivity quantification is needed and thus facilitate the ASO drug development field.

Extracellular vesicles transfer nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander effects.

Ionizing radiation (IR) not only activates DNA damage response (DDR) in irradiated cells but also induces bystander effects (BE) in cells not directly targeted by radiation. How DDR pathways activated in irradiated cells stimulate BE is not well understood. We show here that extracellular vesicles secreted by irradiated cells (EV-IR), but not those from unirradiated controls (EV-C), inhibit colony formation in unirradiated cells by inducing reactive oxygen species (ROS). We found that µEV-IR from Abl nuclear localization signal-mutated ( Abl-µNLS) cells could not induce ROS, but expression of wild-type Abl restored that activity. Because nuclear Abl stimulates miR-34c biogenesis, we measured miR-34c in EV and found that its levels correlated with the ROS-inducing activity of EV. We then showed that EV from miR-34c minigene-transfected, but unirradiated cells induced ROS; and transfection with miR-34c-mimic, without radiation or EV addition, also induced ROS. Furthermore, EV-IR from miR34-family triple-knockout cells could not induce ROS, whereas EV-IR from wild-type cells could cause miR-34c increase and ROS induction in the miR-34 triple-knockout cells. These results establish a novel role for extracellular vesicles in transferring nuclear Abl-dependent and radiation-induced miR-34c into unirradiated cells to cause bystander oxidative stress.