Highlights d ILC2s express the neuropeptide CGRP and its receptor d CGRP inhibits type 2 cytokine production and proliferation of activated ILC2s d Treatment with CGRP negatively regulates IL-33-induced airway inflammation d CGRP receptor-deficient mice show increased ILC2 responses to alarmins

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, poses grave threats to both the global economy and health. The predominant diagnostic screens in use for SARS-CoV-2 detection are molecular techniques such as nucleic acid amplification tests. In this Review, we compare current and emerging isothermal diagnostic methods for COVID-19. We outline the molecular and serological techniques currently being used to detect SARS-CoV-2 infection, past or present, in patients. We also discuss ongoing research on isothermal techniques, CRISPR-mediated detection assays, and point-of-care diagnostics that have potential for use in SARS-CoV-2 detection. Large-scale viral testing during a global pandemic presents unique challenges, chief among them the simultaneous need for testing supplies, durable equipment, and personnel in many regions worldwide, with each of these regions possessing testing needs that vary as the pandemic progresses. The low-cost isothermal technologies described in this Review provide a promising means by which to address these needs and meet the global need for testing of symptomatic individuals as well as provide a possible means for routine testing of asymptomatic individuals, providing a potential means of safely enabling reopenings and early monitoring of outbreaks.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which nucleic acid sequence based amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example, a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform using a cell phone camera module, compatible with field detection.

Isothermal, cell-free, synthetic biology-based approaches to pathogen detection leverage the power of tools available in biological systems, such as highly active polymerases compatible with lyophilization, without the complexity inherent to live-cell systems, of which Nucleic Acid Sequence Based Amplification (NASBA) is well known. Despite the reduced complexity associated with cell-free systems, side reactions are a common characteristic of these systems. As a result, these systems often exhibit false positives from reactions lacking an amplicon. Here we show that the inclusion of a DNA duplex lacking a promoter and unassociated with the amplicon, fully suppresses false positives, enabling a suite of fluorescent aptamers to be used as NASBA tags (Apta-NASBA). Apta-NASBA has a 1 pM detection limit and can provide multiplexed, multicolor fluorescent readout. Furthermore, Apta-NASBA can be performed using a variety of equipment, for example a fluorescence microplate reader, a qPCR instrument, or an ultra-low-cost Raspberry Pi-based 3D-printed detection platform employing a cell phone camera module, compatible with field detection.

Bisphenol-A (BPA), widely used in thermal receipts and food containers, binds to both estrogen receptor (ER) α and β, disrupting estrogen activity and presenting an environmental challenge to the immune system of humans and wildlife. We are exploring the impact of in vivo BPA exposure on zebrafish innate immunity. Zebrafish exposed to 100 ng/ml BPA immediately following fertilization (day 0) show a two to fourfold reduction in gene expression of CXCL8, the chemokine that recruits neutrophils to the site of infection on days 3 through 6 post fertilization (dpf). To determine if reduced chemokine expression has a functional outcome, we studied MPO::GFP zebrafish expressing GFP under a neutrophil-specific myeloperoxidase promoter, which allows tracking of neutrophil movement to a tail cut injury. Compared to controls, 4 dpf BPA-exposed zebrafish show significantly reduced neutrophil recruitment to the site of injury (p=0.004). Control zebrafish receiving a tail cut injury at 4 dpf show a fourfold upregulation in CXCL8 expression one hour post-injury, whereas the BPA-exposed embryos display a blunted CXCL8 response. Because BPA-exposure at 0 dpf leads to a more than twofold increase in CXCR2 expression at 3 and 4 dpf compared to controls, neutrophil migration is likely reduced due to diminished CXCL8. Estradiol is known to reduce CXCL8 expression via the ER, and we found that BPA upregulates ERα and β gene expression by 3 dpf. During the first week of development, control zebrafish show an overall pattern of expression in all four tested genes that differs from that of BPA-exposed embryos. Our findings suggest that BPA leads to the overexpression of ERs, thus reducing CXCL8 expression and diminishing key protective innate immune responses.