Rapid Reversible Osmoregulation of Cytoplasmic Biomolecular Condensates of Human Interferon-α-Induced Antiviral MxA GTPase.

We previously discovered that exogenously expressed GFP-tagged cytoplasmic human myxovirus resistance protein (MxA), a major antiviral effector of Type I and III interferons (IFNs) against several RNA- and DNA-containing viruses, existed in the cytoplasm in phase-separated membraneless biomolecular condensates of varying sizes and shapes with osmotically regulated disassembly and reassembly. In this study we investigated whether cytoplasmic IFN-α-induced endogenous human MxA structures were also biomolecular condensates, displayed hypotonic osmoregulation and the mechanisms involved. Both IFN-α-induced endogenous MxA and exogenously expressed GFP-MxA formed cytoplasmic condensates in A549 lung and Huh7 hepatoma cells which rapidly disassembled within 1-2 min when cells were exposed to 1,6-hexanediol or to hypotonic buffer (~40-50 mOsm). Both reassembled into new structures within 1-2 min of shifting cells to isotonic culture medium (~330 mOsm). Strikingly, MxA condensates in cells continuously exposed to culture medium of moderate hypotonicity (in the range one-fourth, one-third or one-half isotonicity; range 90-175 mOsm) first rapidly disassembled within 1-3 min, and then, in most cells, spontaneously reassembled 7-15 min later into new structures. This spontaneous reassembly was inhibited by 2-deoxyglucose (thus, was ATP-dependent) and by dynasore (thus, required membrane internalization). Indeed, condensate reassembly was preceded by crowding of the cytosolic space by large vacuole-like dilations (VLDs) derived from internalized plasma membrane. Remarkably, the antiviral activity of GFP-MxA against vesicular stomatitis virus survived hypoosmolar disassembly and subsequent reassembly. The data highlight the exquisite osmosensitivity of MxA condensates, and the preservation of antiviral activity in the face of hypotonic stress.

Merging microfluidics with luminescence immunoassays for urgent point-of-care diagnostics of COVID-19.

The Coronavirus disease 2019 (COVID-19) outbreak has urged the establishment of a global-wide rapid diagnostic system. Current widely-used tests for COVID-19 include nucleic acid assays, immunoassays, and radiological imaging. Immunoassays play an irreplaceable role in rapidly diagnosing COVID-19 and monitoring the patients for the assessment of their severity, risks of the immune storm, and prediction of treatment outcomes. Despite of the enormous needs for immunoassays, the widespread use of traditional immunoassay platforms is still limited by high cost and low automation, which are currently not suitable for point-of-care tests (POCTs). Microfluidic chips with the features of low consumption, high throughput, and integration, provide the potential to enable immunoassays for POCTs, especially in remote areas. Meanwhile, luminescence detection can be merged with immunoassays on microfluidic platforms for their good performance in quantification, sensitivity, and specificity. This review introduces both homogenous and heterogenous luminescence immunoassays with various microfluidic platforms. We also summarize the strengths and weaknesses of the categorized methods, highlighting their recent typical progress. Additionally, different microfluidic platforms are described for comparison. The latest advances in combining luminescence immunoassays with microfluidic platforms for POCTs of COVID-19 are further explained with antigens, antibodies, and related cytokines. Finally, challenges and future perspectives were discussed.