Journal: medRxiv

Article Title: SARS-CoV-2 specific immune-signature in direct contacts of COVID-19 cases protect them from contracting disease: A Retrospective Study

doi: 10.1101/2021.03.11.21253367

Figure Lengend Snippet: A , Box plots showing the anti-SARS-CoV-2 IgA, IgM and IgG antibody proportions in serum samples from control, infected and contact along with B , Serum cytokine levels in pg/ml (Eotaxin, G-CSF, IL-7, MIF and MIP 1-□) determined by bio plex. C , The area under the receiver-operating characteristic (ROC) curve of the cytokines having more than 0.8 AUC. D , Box plot depicting the percent inhibition of neutralizing antibodies in an in -vitro spike RBD-ACE2 interaction based Surrogate Virus Neutralization assay. Statistical comparisons were performed using unpaired Wilcoxon test, p-values are written against respective comparisons. Where, n = number of individuals, IgA-Immunoglobulin A, IgM-Immunoglobulin M and IgG-Immunoglobulin G, control (n-14), infected (n-23, Symptomatic n-10 and Asymptomatic n-13) and contact (n-24).

Article Snippet: For IgA and IgM quantification, RBD (SARS-CoV-2 spike RBD recombinant protein, mFc-Tag, CST # 41701S) antigen at a concentration of 200ng/well was coated in a 96-well high binding micro titre plate (HIMEDIA-EP1) in 1x TBS pH-7.4 for 2h at 37□.

Techniques: Infection, Inhibition, In Vitro, Neutralization

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Representative temporal color-coded maps of viral infection time for selected BMDM-fibroblast co-culture conditions (scale bar, 100 μm). The key (far left) demonstrates the color-coding scheme to interpret the remainder of A . Regions infected early during infection are colored darker whereas regions infected later during the experiment are colored lighter. Black regions represent areas without infection. B Quantification of viral spread across all BMDM-fibroblast co-culture conditions. Infection was determined based on fluorescence intensity of tagged viral VP26 in fibroblasts at 48hpi. C Quantification of cells initially infected only from virus (MOI=1) added to the media after being pulsed overnight with different doses of TNF. Infection was determined based on fluorescence intensity of tagged viral VP26 in fibroblasts at 24hpi. D Quantification of cells that stained positive for active, phosphorylated STING (pSTING) after being pulsed overnight with different doses of TNF and infected with MOI 10 of HSV-1. pSTING+ cells were identified based on antibody fluorescence intensity at the specified timepoint post-infection. E Quantification of cells that stained positive for pSTING after being pulsed overnight with different doses of TNF and exposed to dose titrations of the pSTING agonist DMXAA. pSTING+ cells were identified based on antibody fluorescence intensity 1.5 h after DMXAA treatment. Data in B – E are mean ± s.d.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Co-Culture Assay, Fluorescence, Virus, Staining

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Quantification of cell death in NIH 3T3 fibroblasts after treatment with 100 ng/ml TNF or TNF supplemented with 0.25 μg/ml Actinomycin D. Cell death was quantified over time based on Hoechst 33342 fluorescence intensity which increases sharply on nuclear condensation. B Quantification of cell death in fibroblasts 24 h after simultaneous treatment with 0.3 ng/ml TNF and 50 pg/ml LCL-161. Cell death was quantified based on incorporation of a nucleic acid stain (sytox green). C Diagram of experiment for data shown in D . 3T3 fibroblasts were treated for 24 h with 0.3 ng/ml TNF and then washed and rested overnight. The next day, LCL-161 was added at the indicated doses and cell death was quantified after 24 h of LCL-161 exposure. D Quantification of cell death for experiment described in C . Cell death was quantified based on incorporation of sytox green. E Quantification of 3T3 fibroblasts that stained positive for cleaved Caspase-8 during and after treatment with 100 ng/ml TNF. The shaded region indicates the time period before TNF was washed out. Cleaved Caspase-8 was detected by immunostaining and cells were distinguished as positive by setting a threshold on the antibody fluorescence intensity. F Quantification of cell death for 3T3 fibroblasts treated constitutively with 30 ng/ml TNF or washed after overnight treatment with 30 ng/ml TNF. Cell death was quantified based on incorporation of sytox green. G Diagram of experiment for data shown in H . 3T3 fibroblasts were treated with 1 ng/ml TNF for 24 h and then washed. At different times after washing, 0.25 μg/ml Actinomycin D was added to cells for 4 h before quantifying cell death. H Quantification of cell death for experiment described in G . Cell death was quantified based on incorporation of sytox green. B , D , F bars denote the mean of replicates (filled black circles). E , H are mean ± s.d.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Fluorescence, Staining, Immunostaining

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Representative single-cell traces of 3T3 fibroblasts treated with different doses of TNF and infected with MOI 10 of HSV-1. Purple and orange lines represent the fluorescence over time of the virus and nucleic acid stain (sytox green, death marker), respectively. Dotted purple and yellow lines indicate the timing of initial infection and death. The timing of initial infection was determined by setting a threshold on viral VP26 fluorescence intensity. The timing of death was determined by setting a threshold based on incorporation of nucleic acid stain (sytox green) and nuclear condensation identified as a sharp increase in Hoechst 33342 intensity (for details on thresholding, see methods). Shaded regions represent the time interval between infection and death (death decision time, Δt). B Distribution of death decision times obtained by single-cell tracking of 3T3 fibroblasts treated with a single dose of TNF (50 ng/ml) and infected with MOI 10 of HSV-1. Orange line shows an exponential fit of the data. C The same sample that generated B , plotted as the percentage of live 3T3 fibroblasts over time. Orange line shows the same exponential fit plotted in B . D Death rates obtained from exponential fits of 3T3 fibroblasts treated with dose titrations of TNF and infected with MOI 10 of HSV-1. E Quantification of the percentage of uninfected 3T3 fibroblasts killed by TNF (false positives) at 24 h post treatment. Cell death was determined by incorporation of a nucleic acid stain (Sytox green or red), accompanied with nuclear condensation measured as a sharp increase in Hoechst 33342 intensity. F Receiver Operating Characteristic (ROC) curve where false-positive rate represents 3T3 fibroblasts killed by TNF at 24 h post treatment and true positive rate represents infected cells that die during the first viral life cycle (10 h after initial infection is called based on viral VP26 fluorescence intensity). G Speed versus accuracy tradeoff shown by plotting the percentage of false-positive 3T3 fibroblasts (from E ), versus the death rate for infected 3T3 fibroblasts (from D ). D – G are mean ± s.d. F , G colorbar shows TNF concentration.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Fluorescence, Virus, Staining, Marker, Single Cell Tracking, Generated, Concentration Assay

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Diagram of the reactions in our spatial stochastic model. All model parameters were measured in independent experiments shown in Supplementary Fig. , summarized in Supplementary Table . B Ensemble simulation results for different TNF concentrations. Purple area and intensity correspond to the probability of the cell being infected. Black area and intensity correspond to the probability of the cell being dead following infection. Cyan area and intensity correspond to the probability of the cell being alive and healthy. C Quantification of viral spread as a function of TNF concentration, mean ± s.d. D Boxplot summary of simulation results for a dose titration TNF conditions showing the percentages of healthy, infected, dead following infection, and dead but uninfected cells. Central mark denotes the median, boxes denote the upper and lower quartiles, whiskers extend to the most extreme points not considered outliers (≥1.5 interquartile range from top/bottom of box), and outliers are plotted as points. E Quantification of the percentages of healthy, infected, dead following infection, and dead but uninfected 3T3 fibroblasts treated with dose titrations of TNF and infected with MOI 1 of HSV-1. Data are mean ± s.d. F Simulation of the dynamics of actively infected cells for different concentrations of TNF. Lines represent the mean values of 500 simulations. G Effective reproductive rates (Rt) distributions in different conditions represented as violin plots, which were generated with non-negative support and a bandwidth of 0.05. In this context, Rt represents the ratio of the probability that a cell is infected by its infected neighbor to the probability of that neighboring cell dying. The red line denotes a separatrix: Above it, the virus is more likely to spread further and below it, the virus is more likely to be extinguished. Black lines indicate median. For simulations ( B – D , F , G ), each condition was simulated 500 times.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Concentration Assay, Titration, Generated, Virus

Journal: Nature Communications

Article Title: TNF controls a speed-accuracy tradeoff in the cell death decision to restrict viral spread

doi: 10.1038/s41467-021-23195-9

Figure Lengend Snippet: A Cartoon diagram with cornea emphasized to show different perspectives of images. B Representative transverse image of R26-H2B-mCherry mouse corneas 36 h after infection with HSV-1 and treated with either 0 or 50 ng/ml TNF. C Enlarged regions within images shown in B with cell death channel included. D Quantification of the total sum of virus pixels for C57Bl/6 corneas treated with 0 or 50 ng/ml TNF, infected with HSV-1, and imaged at 48hpi. E Percentage of total virus pixels also positive for Sytox Green death marker. F Representative epithelial projections of C57Bl/6 corneas treated with 0 or 50 ng/ml TNF and imaged 24 h after treatment. G Quantification of the total Sytox Green intensity for uninfected corneas. H Speed versus accuracy tradeoff shown by plotting the percentage of false-positive primary corneal epithelial cells versus the death rate for infected primary corneal epithelial cells. Colorbar represents TNF concentration. Data are mean ± s.d. D , E , G bars denote the mean of replicate eyes (filled black circles). All scale bars, 100 μm.

Article Snippet: 12.5 × 10 4 3T3 cells were seeded per well of black 96-well plates in imaging media (Fluorobrite with 10% NBCS, 2 mM L-glutamine, 10U/ml Penicillin and 10 μg/ml Streptomycin) supplemented, where indicated, with dose titrations of recombinant mouse TNFα (Cell Signaling Technologies), 30 μM Z-VAD(OMe)-FMK (Cayman Chemical), 30 μM Necrostatin-1 (Sigma–Aldrich), 0.25 μg/ml Actinomycin D (Thermo Fisher), or the specified doses of LCL-161 (Cayman Chemical).

Techniques: Infection, Virus, Marker, Concentration Assay