Two proteins, STIM1 in the endoplasmic reticulum and Orai1 in the plasma membrane, are required for the activation of Ca 2؉ releaseactivated Ca 2؉ (CRAC) channels at the cell surface. How these proteins interact to assemble functional CRAC channels has remained uncertain. Here, we determine how many Orai1 and STIM1 molecules are required to form a functional CRAC channel. We engineered several genetically expressed fluorescent Orai1 tandem multimers and a fluorescent, constitutively active STIM1 mutant. The tandem multimers assembled into CRAC channels, as seen by rectifying inward currents and by cytoplasmic calcium elevations. CRAC channels were visualized as fluorescent puncta in total internal reflection microscopy. With single-molecule imaging techniques, it was possible to observe photo-bleaching of individual fluorophores and to count the steps of bleaching as a measure of the stoichiometry of each CRAC channel complex. We conclude that the subunit stoichiometry in an active CRAC channel is four Orai1 molecules and two STIM1 molecules. Fluorescence resonance energy transfer experiments also showed that four Orai1 subunits form the assembled channel. From the fluorescence intensity of single fluorophores, we could estimate that our transfected HEK293 cells had almost 400,000 CRAC channels and that, when intracellular Ca 2؉ stores were depleted, the channels clustered in aggregates containing Ϸ1,300 channels, amplifying the local Ca 2؉ entry.endoplasmic reticulum ͉ fluorescence resonance energy transfer

With COVID-19 spreading around the world, many countries are exposed to the imported case risk from inbound international flights. Several governments issued restrictions on inbound flights to mitigate such risk. But with the pandemic controlled in many countries, some decide to reopen the economy by relaxing the international air travel bans. As the virus has still been prevailing in many regions, this relaxation raises the alarm to import overseas cases and results in the revival of local pandemic. This study proposes a risk index to measure one country's imported case risk from inbound international flights. The index combines both daily dynamic international air connectivity data and the updated global COVID-19 data. It can measure the risk at the country, province and even specific route level. The proposed index was applied to China, which is the first country to experience and control COVID-19 pandemic while later becoming exposed to high imported case risk after the epidemic centers switched to Europe and the US afterward. The calculated risk indexes for each Chinese province or region show both spatial and temporal patterns from January to April 2020. It is found that China's strict restriction on inbound flights since March 26 was very effective to cut the imported case risk by half than doing nothing. But the overall index level kept rising because of the deteriorating pandemic conditions around the world. Hong Kong and Taiwan are the regions facing the highest imported case risk due to their superior international air connectivity and looser restriction on inbound flights. Shandong Province had the highest risk in February and early March due to its well-developed air connectivity with South Korea and Japan when the pandemic peaked in these two countries. Since mid-March, the imported case risk from Europe and the US dramatically increased. Last, we discuss policy implications for the relevant stakeholders to use our index to dynamically adjust the international air travel restrictions. This risk index can also be applied to other contexts and countries to relax restrictions on particular low-risk routes while still restricting the high-risk ones. This would balance the essential air travels need and the requirement to minimize the imported case risk.

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