Journal: Research Square

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.21203/rs.3.rs-802084/v1

Figure Lengend Snippet: Higher anti-RBD IgM seroconversion rate in respiratory samples compared to paired blood samples of COVID-19 patients. a ELISA titration curves against the SARS-CoV-2 receptor-binding domain (RBD) for IgM, IgG, and IgA in COVID-19 respiratory and paired blood samples and non-COVID-19 respiratory samples. Dotted lines within each graph indicates the cut-off used to determine end-point titres. b Endpoint titres of SARS-CoV-2 RBD antibodies between (i) respiratory samples of COVID-19 and non-COVID-19 patients, and (ii) plasma and respiratory samples of COVID-19 patients. (i) Bars indicate median with interquartile range. Dotted line indicates the detection level. (ii) Dotted lines connect the most closely matched plasma and respiratory samples from each patient. Statistical significance was determined with Mann-Whitney test. c ELISA titration curves against the SARS-CoV-2 RBD for 3 COVID-19 patients with serial respiratory samples. d Heatmap of percentage (%) inhibition tested by surrogate virus neutralization test (sVNT) and anti-RBD ELISA titres. e Correlation between anti-RBD antibody titres and (%) sVNT inhibition. Correlation was determined with Spearman’s correlation. f Number of (i) samples and (ii) patients with seroconverted anti-RBD IgM, IgG, IgA and positive % sVNT inhibition. Pink curved lines surrounding the donut graphs indicate the samples/patients with seroconverted IgM. Earliest samples were used for each patient when determining seroconversion which was defined as average titre +2xSD of non-COVID-19 samples. Positive % sVNT inhibition was defined as % sVNT inhibition ≥ 20%.

Article Snippet: DuoSet ELISA ancillary reagent kit (R&D Systems) was used for respiratory fluids and in-house reagents with the same composition were used for plasma samples.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Binding Assay, Clinical Proteomics, MANN-WHITNEY, Inhibition, Virus, Neutralization

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Response function of Orai1-GCaMP6f. (A and B) Residual plots for nonlinear curve fitting to the Hill equation (A; R 2 = 0.992) and linear regression (B; R 2 = 0.978) for the scatterplots of whole-cell current versus fluorescence shown in . (C and D) Scatterplots of normalized whole-cell current versus fluorescence for steps to increasingly negative membrane potentials (−10, −40, −70, and −100 mV; cells A, E, and F; red diamonds) and for individual test pulses to negative membrane potentials (−10, −40, −70, and −100 mV; cells A–F; blue diamonds). Lines indicate fitting to the Hill equation for a stepped test pulse (light red; R 2 = 0.993, hc = 1.7 with 95% CI of 1.3–2.3) and for individual test pulses (light blue; R 2 = 0.957, hc = 1.7 with 95% CI of 1.1–2.7). Note the high degree of overlap with similar maximum fluorescence values for the two fitted curves. (D) Enlargement of lower central portion of C. Whole-cell recordings were performed in 2 mM external Ca 2+ .

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Fluorescence, Membrane

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Biophysical properties of Orai1-GCaMP6f and GFP-Orai1 STIM1-operated currents. Representative whole-cell recordings from HEK 293A cells transiently cotransfected at a ratio of 10:1 with mCherry-STIM1 plus either Orai1-GCaMP6f (A–D, top; n = 6 cells) or GFP-Orai1 (E–H, bottom; n = 6 cells). (A and E) Leak-subtracted time course, following break-in to achieve whole-cell recording at time = 0, of Orai1-GCaMP6f (A) and GFP-Orai1 (E) STIM1-operated current recorded in response to ramp stimuli from −120 mV (black points) to +100 mV (gray points). (B, C, F, and G) Current-voltage relationship of Orai1-GCaMP6f (B and C) and GFP-Orai1 (F and G) current recorded in 20 mM Ca 2+ (B and F) and Na + DVF (C and G) solutions, respectively. (D and H) Ca 2+ -dependent inactivation of Orai1-GCaMP6f (D) and GFP-Orai1 (H) current during −120 mV hyperpolarizing pulse from a holding potential of 0 mV, recorded in 20 mM Ca 2+ external solution. Bars indicate exchange of external solutions (see for solution recipes). Arrows in A and D indicate the time points for I-V curves in B, C, F, and G. Lines with arrowheads indicate biophysical parameters presented in .

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques:

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Orai1-GCaMP6f fluorescence responses to changes in membrane potential during whole-cell recording and Ca 2+ perfusion after cell unroofing. (A and F) TIRF micrographs of cotransfected HEK 293A cells showing Orai1-GCaMP6f (A) and mCherry-STIM1 (F) before pipette break-in. The image intensities have been increased threefold with respect to B–E and G–J to facilitate comparison. (B, C, G, and H) TIRF images of Orai1-GCaMP6f (B) and mCherry-STIM1 (G) at +20 mV holding potential and during −100 mV test pulse (C and H). (D, E, I, and J) TIRF images of the same cell after mechanical unroofing showing Orai1-GCaMP6f and mCherry-STIM1 upon perfusion of solutions containing (D and I) and lacking (E and J) 2 mM Ca 2+ . Scale bar in J is 20 µm (applies to A–I). (K–M) Plots of whole-cell current (K), Orai1-GCaMP6f fluorescence (L), and mCherry-STIM1 (M) fluorescence in response to test pulses to −40 and −100 mV. Note the parallel changes in Orai1-GCaMP6f current and fluorescence during test pulses and the lack of change in mCherry-STIM1 fluorescence. (N) Comparison of Orai1-GCaMP6f fluorescence at +20 mV holding potential and upon −40 and −100 mV test pulses with Orai1-GCaMP6f fluorescence from the same, subsequently unroofed, cell perfused with solutions containing (2 Ca) and lacking (0 Ca) 2 mM Ca 2+ . Data are representative of six cells acquired over 4 d. Images and traces correspond to cell E (see ).

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Fluorescence, Membrane, Transferring, Comparison

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Measurement of Orai1-GECI affinity and kinetics. HEK 293A cells were cotransfected with Orai1-GCaMP6f and mCherry-STIM1, or with Orai1-G-GECO 1.2 and mCherry-STIM1. ER Ca 2+ stores were depleted by whole-cell break-in and dialysis with pipette solutions containing IP 3 . (A–D) Unroofed cells were sequentially perfused with buffered Ca 2+ solutions in the order indicated and imaged by TIRF microscopy. (A and B) Orai1-GCaMP6f in situ binding affinity. (A) Normalized Orai1-GCaMP6f fluorescence from the cell footprint. Note the lack of photobleaching of Orai1-GCaMP6f. (B) Corresponding association curve, fitted by the Hill equation with K d of 620 nM and an hc of 1.56 (gray line; R 2 = 0.993). 95% CIs were 590–660 nM for the K d and 1.44–1.68 for the hc. n = 7 cells acquired over 3 d. (C and D) Orai1-G-GECO 1.2 in situ binding affinity. Normalized Orai1-G-GECO 1.2 fluorescence from the cell footprint (C) and association curve (D), fitted by the Hill equation with K d of 1,010 nM and hc of 1.88 (gray line; R 2 = 0.994). 95% CIs were 950–1,070 nM for the K d and 1.74–2.05 for the hc. n = 6 cells acquired over 2 d. (E–G) Orai1-GCaMP6f fluorescence rise and fall kinetics during whole-cell recording. n = 6 cells. (E) Rising phase of Orai1-GCaMP6f fluorescence in response to a 600-ms test pulse to −100 mV in 2 mM extracellular Ca 2+ . The gray line, representing the time course of Ca 2+ association with the tethered GCaMP6f probe, indicates a fit to the sum of a single exponential association with a time constant of 71 ms and a linear rising function (R 2 = 0.949). The red line indicates the linear component of the rising phase. Half-rise time was 49 ms (43–56 ms, 95% CI). (F) Mean rising phase Orai1-GCaMP6f fluorescence of HEK 293A cells in response to a 600-ms test pulse to −100 mV in 20 mM extracellular Ca 2+ . Gray line indicates the fit to a single exponential association function with a time constant of 33 ms (R 2 = 0.961). (G) Mean falling phase of Orai1-GCaMP6f fluorescence of HEK 293A cells upon stepping back to +20 mV from −100 mV in 2 mM extracellular Ca 2+ . Gray line indicates a single exponential decay function with a time constant of 62 ms (R 2 = 0.989). Half-fall time was 43 ms (41–45 ms, 95% CI). Error bars are ± SEM.

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Transferring, Microscopy, In Situ, Binding Assay, Fluorescence

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Orai1-GCaMP6f current and fluorescence in response to changes in membrane potential and external Ca 2+ . (A and B) Whole-cell currents (A) and Orai1-GCaMP6f fluorescence (B) in response to a series of steps to increasingly negative membrane potential in 2 mM external Ca 2+ . Membrane potentials are indicated above the traces. Data from three different cells. (C and D) Plots of Orai1-GCaMP6f whole-cell current (C) and fluorescence (D) for the same three cells as a function of test potential in 2 mM external Ca 2+ . (E and F) Scatterplots of whole-cell current versus fluorescence (colored diamonds) for the same three cells with fitting to the Hill equation (gray line; R 2 = 0.992 and hc = 1.6 with 95% CI of 1.2–2.2). (F) Enlargement of lower central portion of E. (G and H) Plots of Orai1-GCaMP6f whole-cell current (G) and fluorescence (H) in response to a series of 600 ms duration −100 mV test pulses at increasing external Ca 2+ concentrations. External Ca 2+ concentration in mM are indicated beside the traces. G and H correspond to cell E (see ). AU, arbitrary unit.

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Fluorescence, Membrane, Concentration Assay

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Correspondence between puncta ΔF/F0, A-index, and relative STIM1/Orai1 ratio. Maps of plateau ΔF/F0 (A–E), A-index (F–J), and relative STIM1/Orai1 ratio (K–O) for five HEK 293A cells cotransfected with Orai1-GCaMP6f and mCherry-STIM1 in response to a 600-ms test pulse to −100 mV in 2 mM extracellular Ca 2+ . White arrowheads indicate puncta with little or no change in fluorescence in the corners of the cell footprint, and orange arrowheads indicate puncta with and low A-indices. Note that puncta in all seven corners of the cell footprint in J exhibit slow activation of Orai1-GCaMP6f fluorescence. Scale bar in A is 20 µm (applies to B–O). A, F, and K correspond to cell A; B, G, and L to cell C; C, H, and M to cell D; D, I, and N to cell E; and E, J, and O to cell F (see ).

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Fluorescence, Activation Assay

Journal: The Journal of General Physiology

Article Title: Cell-wide mapping of Orai1 channel activity reveals functional heterogeneity in STIM1-Orai1 puncta

doi: 10.1085/jgp.201812239

Figure Lengend Snippet: Measurement of current through individual puncta. (A) Plot of the plateau fractional saturation of Orai1-GCaMP6f at puncta in cell E during a 600-ms test pulse to −100 mV. Red line indicates the mean. (B) Map of Orai1-GCaMP6f fractional activation at puncta. Light gray line indicates the edge of the cell footprint. (C) Enlargement of central right portion of B. (D) Plot of plateau [Ca 2+ ] at puncta in cell E. Red line indicates the median. (E) Map of plateau [Ca 2+ ] at puncta. (F) Enlargement of central right portion of E. (G) Plot of Orai1 plateau Ca 2+ current through puncta in cell E. Red line indicates the median. (H) Map of Orai1 plateau Ca 2+ current at puncta. (I) Enlargement of central right portion of H. (J) Scatterplot of ΔF/F0 versus resting Orai1-GCaMP6f fluorescence intensity (F0) at individual puncta for all six cells. Puncta STIM1:Orai1 ratio was restricted to values between 2 and 3. Linear curve fit (red line) indicates that puncta ΔF/F0 is not related to the number of Orai1-GCaMP6f channels at a punctum (slope = −0.00001, R 2 = 0.00002; slope ≠ 0, P = 0.95, F test). Scale bar in B is 10 µm (applies to E and H), and the scale bar in C is 2.5 µm (applies to F and I).

Article Snippet: Standard buffered Ca 2+ solutions (Calcium Calibration Buffer Kit #1; Thermo Fisher Scientific) with K + as the primary cation and varying free Ca 2+ concentrations were used in probe calibration runs.

Techniques: Activation Assay, Fluorescence

Journal: Journal of Medical Devices

Article Title: Advances in Diagnostic Methods for Zika Virus Infection

doi: 10.1115/1.4041086

Figure Lengend Snippet: Currently authorized ZIKV diagnostic tests under emergency use authorization

Article Snippet: Refer to Table for currently FDA approved ZIKV diagnostic methods. table ft1 table-wrap mode="anchored" t5 Table 1 caption a7 Name of the Kit Organization Sample Type Volume Sensitivity Specificity Process Time Point of Care Nucleic assay based assays: Aptima Zika Virus Assay [ 39 ] Hologic, Inc. Serum, Plasma and Urine Serum and Plasma: 1200 μ L Serum and Plasma: 100% Serum and Plasma: 97.2% 3.5 h No Urine: 2000 μ L Urine: 100% Urine: 100% Gene-RADAR Zika virus test [ 40 ] Nanobiosym Diagnostics, Inc. Serum Serum: 50 μ L 100% 100% 1 h No RealStar® Zika virus RT–PCR Kit 1.1 [ 41 ] Altona Diagnostics Serum and Urine Serum and Urine: 140 μ L 100% 96.6% 2 h No Realtime Zika [ 42 ] Abbott Molecular, Inc.

Techniques: Diagnostic Assay, Quantitative RT-PCR, Luminex, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay