Investigating transmission of SARS-CoV-2 using novel face mask sampling: a protocol for an observational prospective study of index cases and their contacts in a congregate setting.

INTRODUCTION: This study aims to measure how transmission of SARS-CoV-2 occurs in communities and to identify conditions that lend to increased transmission focusing on congregate situations. We will measure SARS-CoV-2 in exhaled breath of asymptomatic and symptomatic persons using face mask sampling-a non-invasive method for SARS-CoV-2 detection in exhaled air. We aim to detect transmission clusters and identify risk factors for SARS-CoV-2 transmission in presymptomatic, asymptomatic and symptomatic individuals. METHODS AND ANALYSIS: In this observational prospective study with daily follow-up, index cases and their respective contacts are identified at each participating institution. Contact definitions are based on Centers for Disease Control and Prevention and local health department guidelines. Participants will wear masks with polyvinyl alcohol test strips adhered to the inside for 2 hours daily. The strips are applied to all masks used over at least 7 days. In addition, self-administered nasal swabs and (optional) finger prick blood samples are performed by participants. Samples are tested by standard PCR protocols and by novel antigen tests. ETHICS AND DISSEMINATION: This study was approved by the Colorado Multiple Institutional Review Board and the WHO Ethics Review Committee. From the data generated, we will analyse transmission clusters and risk factors for transmission of SARS-CoV-2 in congregate settings. The kinetics of asymptomatic transmission and the evaluation of non-invasive tools for detection of transmissibility are of crucial importance for the development of more targeted control interventions-and ultimately to assist with keeping congregate settings open that are essential for our social fabric. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov (#NCT05145803).

A genetically encoded probe for imaging nascent and mature HA-tagged proteins in vivo.

To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.

Differential Desensitization Observed at Multiple Effectors of Somatic µ-Opioid Receptors Underlies Sustained Agonist-Mediated Inhibition of Proopiomelanocortin Neuron Activity.

Activation of somatic µ-opioid receptors (MORs) in hypothalamic proopiomelanocortin (POMC) neurons leads to the activation of G-protein-coupled inward rectifier potassium (GIRK) channels and hyperpolarization, but in response to continued signaling MORs undergo acute desensitization resulting in robust reduction in the peak GIRK current after minutes of agonist exposure. We hypothesized that the attenuation of the GIRK current would lead to a recovery of neuronal excitability whereby desensitization of the receptor would lead to a new steady state of POMC neuron activity reflecting the sustained GIRK current observed after the initial decline from peak with continued agonist exposure. However, electrophysiologic recordings and GCaMP6f Ca2+ imaging in POMC neurons in mouse brain slices indicate that maximal inhibition of cellular activity by these measures can be maintained after the GIRK current declines. Blockade of the GIRK current by Ba2+ or Tertiapin-Q did not disrupt the sustained inhibition of Ca2+ transients in the continued presence of agonist, indicating the activation of an effector other than GIRK channels. Use of an irreversible MOR antagonist and Furchgott analysis revealed a low receptor reserve for the activation of GIRK channels but a >90% receptor reserve for the inhibition of Ca2+ events. Altogether, the data show that somatodendritic MORs in POMC neurons inhibit neuronal activity through at least two effectors with distinct levels of receptor reserve and that differentially reflect receptor desensitization. Thus, in POMC cells, the decline in the GIRK current during prolonged MOR agonist exposure does not reflect an increase in cellular activity as expected.SIGNIFICANCE STATEMENT Desensitization of the µ-opioid receptor (MOR) is thought to underlie the development of cellular tolerance to opiate therapy. The present studies focused on MOR desensitization in hypothalamic proopiomelanocortin (POMC) neurons as these neurons produce the endogenous opioid ß-endorphin and are heavily regulated by opioids. Prolonged activation of somatic MORs in POMC neurons robustly inhibited action potential firing and Ca2+ activity despite desensitization of the MOR and reduced activation of a potassium current over the same time course. The data show that somatic MORs in POMC neurons couple to multiple effectors that have differential sensitivity to desensitization of the receptor. Thus, in these cells, the cellular consequence of MOR desensitization cannot be defined by the activity of a single effector system.

Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER-plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER-plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER-plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER-plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca(2+) signaling.

Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

Regulation of Kv2.1 K(+) conductance by cell surface channel density.

The Kv2.1 voltage-gated K(+) channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites, and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are nonconducting. Using total internal reflection fluorescence microscopy, the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared with K(+) channel conductance measured by whole-cell voltage clamp of the same cell. This approach indicated that, as channel density increases, nonclustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the nonconducting state with 17% conducting K(+) at higher surface densities. The nonconducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immunofluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared with the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 d, respectively. Together, these data indicate that the nonconducting state depends primarily on surface density as opposed to cluster location and that this nonconducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K(+) conductance further supports a nonconducting role for Kv2.1 in excitable tissues.

Size of cell-surface Kv2.1 domains is governed by growth fluctuations.

The Kv2.1 voltage-gated potassium channel forms stable clusters on the surface of different mammalian cells. Even though these cell-surface structures have been observed for almost a decade, little is known about the mechanism by which cells maintain them. We measure the distribution of domain sizes to study the kinetics of their growth. Using a Fokker-Planck formalism, we find no evidence for a feedback mechanism present to maintain specific domain radii. Instead, the size of Kv2.1 clusters is consistent with a model where domain size is established by fluctuations in the trafficking machinery. These results are further validated using likelihood and Akaike weights to select the best model for the kinetics of domain growth consistent with our experimental data.