Platform Incubator with Movable XY Stage: A New Platform for Implementing In-Cell Fast Photochemical Oxidation of Proteins.

Fast Photochemical Oxidation of proteins (FPOP) coupled with mass spectrometry (MS) has become an invaluable tool in structural proteomics to interrogate protein interactions, structure, and protein conformational dynamics as a function of solvent accessibility. In recent years, the scope of FPOP, a hydroxyl radical protein foot printing (HRPF) technique, has been expanded to protein labeling in live cell cultures, providing the means to study protein interactions in the convoluted cellular environment. In-cell protein modifications can provide insight into ligand induced structural changes or conformational changes accompanying protein complex formation, all within the cellular context. Protein footprinting has been accomplished employing a customary flow-based system and a 248 nm KrF excimer laser to yield hydroxyl radicals via photolysis of hydrogen peroxide, requiring 20 minutes of analysis for one cell sample.To facilitate time-resolved FPOP experiments, the use of a new 6-well plate-based IC-FPOP platform was pioneered. In the current system, a single laser pulse irradiates one entire well, which truncates the FPOP experimental time frame resulting in 20 seconds of analysis time, a 60-fold decrease. This greatly reduced analysis time makes it possible to research cellular mechanisms such as biochemical signaling cascades, protein folding, and differential experiments (i.e., drug-free vs. drug bound) in a time-dependent manner. This new instrumentation, entitled Platform Incubator with Movable XY Stage (PIXY), allows the user to perform cell culture and IC-FPOP directly on the optical bench using a platform incubator with temperature, CO2 and humidity control. The platform also includes a positioning stage, peristaltic pumps, and mirror optics for laser beam guidance. IC-FPOP conditions such as optics configuration, flow rates, transient transfections, and H2O2 concentration in PIXY have been optimized and peer-reviewed. Automation of all components of the system will reduce human manipulation and increase throughput.

Rapid and low-cost sampling for detection of airborne SARS-CoV-2 in dehumidifier condensate.

Airborne spread of coronavirus disease 2019 (COVID-19) by infectious aerosol is all but certain. However, easily implemented approaches to assess the actual environmental threat are currently unavailable. We present a simple approach with the potential to rapidly provide information about the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the atmosphere at any location. We used a portable dehumidifier as a readily available and affordable tool to collect airborne virus in the condensate. The dehumidifiers were deployed in selected locations of a hospital ward with patients reporting flu-like symptoms which could possibly be due to COVID-19 over three separate periods of one week. Samples were analyzed frequently for both virus envelope protein and SARS-CoV-2 RNA. In several samples across separate deployments, condensate from dehumidifiers tested positive for the presence of SARS-CoV-2 antigens as confirmed using two independent assays. RNA was detected, but not attributable to SARS-CoV-2. We verified the ability of the dehumidifier to rapidly collect aerosolized sodium chloride. Our results point to a facile pool testing method to sample air in any location in the world and assess the presence and concentration of an infectious agent to obtain quantitative risk assessment of exposure, designate zones as "hot spots" and minimize the need for individual testing which may often be time consuming, expensive, and laborious.

Manufacturing biological medicines on demand: Safety and efficacy of granulocyte colony-stimulating factor in a mouse model of total body irradiation.

Protein therapeutics, also known as biologics, are currently manufactured at centralized facilities according to rigorous protocols. The manufacturing process takes months and the delivery of the biological products needs a cold chain. This makes it less responsive to rapid changes in demand. Here, we report on technology application for on-demand biologics manufacturing (Bio-MOD) that can produce safe and effective biologics from cell-free systems at the point of care without the current challenges of long-term storage and cold-chain delivery. The objective of the current study is to establish proof-of-concept safety and efficacy of Bio-MOD-manufactured granulocyte colony-stimulating factor (G-CSF) in a mouse model of total body irradiation at a dose estimated to induce 30% lethality within the first 30 days postexposure. To illustrate on-demand Bio-MOD production feasibility, histidine-tagged G-CSF was manufactured daily under good manufacturing practice-like conditions prior to administration over a 16-day period. Bio-MOD-manufactured G-CSF improved 30-day survival when compared with saline alone (p = .073). In addition to accelerating recovery from neutropenia, the platelet and hemoglobin nadirs were significantly higher in G-CSF-treated animals compared with saline-treated animals (p < .05). The results of this study demonstrate the feasibility of consistently manufacturing safe and effective on-demand biologics suitable for real-time release.

Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage.

Fast photochemical oxidation of proteins (FPOP) is a protein footprinting technique that is being increasingly used in MS-based proteomics. FPOP is utilized to study protein-protein interactions, protein-ligand interactions, and protein conformational dynamics. This method has recently been extended to protein labeling in live cells (IC-FPOP), allowing the study of protein conformations in the complex cellular environment. Traditionally, IC-FPOP has been executed using a single cell flow system, in which hydrodynamic focusing drives cells along in a single file line, keeping the cells from clumping and thus ensuring equal exposure to the laser irradiation required for photochemical oxidation. Here, we introduce a novel platform that allows IC-FPOP to occur in a sterile incubation system complete with a mobile stage for XY movement, peristaltic pumps equipped with perfusion lines for chemical transport, and mirrors for laser beam guidance. This new system, called Platform Incubator with movable XY stage (PIXY), also utilizes software enabling automated communication between equipment and execution of the entire system. Further, comparison with a standard IC-FPOP flow system results reveal that this platform can successfully be used in lieu of the flow system while also decreasing the time to complete analysis of a single sample.

Point-of-care production of therapeutic proteins of good-manufacturing-practice quality.

Manufacturing technologies for biologics rely on large, centralized, good-manufacturing-practice (GMP) production facilities and on a cumbersome product-distribution network. Here, we report the development of an automated and portable medicines-on-demand device that enables consistent, small-scale GMP manufacturing of therapeutic-grade biologics on a timescale of hours. The device couples the in vitro translation of target proteins from ribosomal DNA, using extracts from reconstituted lyophilized Chinese hamster ovary cells, with the continuous purification of the proteins. We used the device to reproducibly manufacture His-tagged granulocyte-colony stimulating factor, erythropoietin, glucose-binding protein and diphtheria toxoid DT5. Medicines-on-demand technology may enable the rapid manufacturing of biologics at the point of care.