Mean ± SEM or mean ± SD were determined for continuous variables as noted. Technical and biological replicates are described in the figure legends. For analysis of mouse studies, the comparison of weight-change curves was performed using a one-way ANOVA with Dunnett’s post hoc test using Prism v.9.0 (GraphPad). Viral burden and gene-expression measurements were compared to each other or the isotype control using a one-way ANOVA with Tukey’s or Dunnett’s post hoc test, respectively, using Prism v.9.0 (GraphPad). Survival curves were estimated using the Kaplan-Meier method and differences assessed using the log-rank Mantel-Cox test and a Bonferroni correction for multiple comparisons using Prism v.9.0 (GraphPad).
Prism v9
Manufactured by GraphPad
Sourced in United States, Austria, Japan, Finland, Belgium
Prism v9 is a data analysis and graphing software developed by GraphPad. It provides tools for curve fitting, statistical analysis, and creating publication-quality graphs and figures.
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Lab products found in correlation
Purelink rna mini kit, by Thermo Fisher Scientific (39 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (38 mentions)
Sas v9, by SAS Institute (25 mentions)
Ds 11 spectrophotometer, by DeNovix (19 mentions)
Spss v26, by IBM (16 mentions)
Spss v25, by IBM (13 mentions)
Spss v27, by IBM (11 mentions)
Sas version 9, by SAS Institute (10 mentions)
Spss v22, by IBM (7 mentions)
Stata v17, by StataCorp (6 mentions)
Spss v23, by IBM (6 mentions)
Spss v28, by IBM (6 mentions)
Stata 16, by StataCorp (5 mentions)
Prism 9, by GraphPad (5 mentions)
R version 4, by R Project for Statistical Computing (5 mentions)
Spss v24, by IBM (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Jmp pro, by SAS Institute (4 mentions)
Spss statistics v25, by IBM (4 mentions)
2 686 protocols using prism v9
1
Mouse Weight, Viral Burden, and Survival
2
Anti-DEspR Neutrophil Modulation in LPS-Induced Models
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Descriptive statistics were obtained and the appropriate statistical tests were applied for the different studies. Kruskal-Wallis with Dunn’s multiple comparison testing was performed to analyze differences in BALF neutrophils among study groups in human volunteers. Paired t-test was used to assess differences in peripheral neutrophil counts and NLR pre- and post-LPS segmental challenge (GraphPad Prism V9.4). For the LPS-ALI macaque study comparing saline mock-treated control vs antibody-treated groups in different parameters across a 24-72-hour time course, we used One Way Repeated Measures ANOVA followed by the Holm-Sidak test for multiple comparisons (SigmaPlot 11.0). For group contingency analysis of neutrophil adhesion and effects of anti-DEspR treatment on DEspR+ macaque neutrophil adhesion, we used Fisher’s Exact test (GraphPad Prism V9.4). For the LPS-encephalopathy rat model survival studies, we used Kaplan-Meier survival curves testing for statistical significance using the Mantel Cox Log Rank Sum test (GraphPad Prism V9.4). For in vitro assays of anti-DEspR effects on rat neutrophil survival, and analysis of anti-DEspR antibody target engagement and bioeffects we used One Way ANOVA followed by Holm-Sidak test for multiple comparisons (SigmaPlot 11.0). P < 0.05 was considered statistically significant.
3
Anti-SARS-CoV-2 Neutralization Assays
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The anti-SARS-CoV-2 analyses were done at least in triplicate. Plant hot water extracts had n ≥ 6 independent assays as previously described (Nair et al., 2021 ). IC50 values were calculated using GraphPad Prism V9.4. The endpoint titer of each replicate was scored and plotted using GraphPad Prism V9.4 and statistics using t-test (Wilcoxon matched-pairs signed rank test) was performed between the groups to determine the p-value. The percentage neutralization was calculated and plotted using GraphPad Prism V9.4 software.
4
Comparative Statistical Analysis of Bacterial Loads
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Statistical comparison of bacterial loads between lines was performed by one-way ANOVA using GraphPad Prism v9.0 (La Jolla, CA). Quantitative PCR bacterial load data were log10-transformed prior to statistical analyses. Two ARS-Fp-R line fish had mean values below the average qPCR limit of quantification (0.5 GE/100 ng DNA, Ct>36.8) and were not included in the load comparison. All statistics were run with a significance level of p < 0.05.
Biomarker values were compared by ANOVA using a Kruskal–Wallis test with Dunn’s multiple comparisons calculated using GraphPad Prism v9.0. Linear regression, 95% confidence intervals, and Spearman correlations were calculated using GraphPad Prism v9.0. Principal component analysis, heat maps, hierarchal clustering, and network analyses were performed using Qlucore Omics Explorer v3.8 (Qlucore, Lund Sweden).
Biomarker values were compared by ANOVA using a Kruskal–Wallis test with Dunn’s multiple comparisons calculated using GraphPad Prism v9.0. Linear regression, 95% confidence intervals, and Spearman correlations were calculated using GraphPad Prism v9.0. Principal component analysis, heat maps, hierarchal clustering, and network analyses were performed using Qlucore Omics Explorer v3.8 (Qlucore, Lund Sweden).
5
Kaplan-Meier and Cox Regression Analysis
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Kaplan–Meier curves were generated with Prismv9.40 (GraphPad, San Diego, CA, USA) and analyzed with a log-rank test. Unpaired t-tests and ANOVA analyses were performed with Prismv9.40 (GraphPad). Cox proportional hazards models were calculated with JMPv14.0.0 (SAS, Stockholm, Sweden).
6
Statistical Analyses for CPFE Studies
7
Quantitative Fluorescent EMSA Analysis
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20 μl reactions contained EMSA buffer (50 mM Tris–HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 5 mM MgCl2, 4% (v/v) glycerol, 0.025 mg/ml BSA) with 10 nM fluorescein-labelled DNA and 2-fold serial dilutions of protein, with maximum protein concentration as indicated in the respective figures. Reactions were incubated at 30°C for 20 min and resolved on an 8% native PAGE or 4–20% gradient PAGE gel. Electrophoresis was performed in 0.5X TBE buffer at room temperature (21°C). Experiments (n = 3 technical replicates at minimum) were imaged with an Amersham Typhoon Imager (GE Healthcare) and analysed with ImageJ by quantifying the intensity of free DNA as protein concentration increased (28 (link)). DNA binding curves were plotted as a function of the fraction of DNA bound compared to an unbound DNA control. Dissociation constants were obtained through calculation of specific binding with Hill slope in Prism v.9.0 (GraphPad). p-values were calculated by a two-tailed Welch's t-test in Prism v.9.0 (GraphPad).
8
Kinetics of DNA Ligation Assay
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The ligation assay was adapted from a previously published protocol (30–32 (link)). Briefly, 20 μl ligation reaction mixtures containing 50 mM HEPES pH 8.0, 5 mM DTT, 5 mM MgCl2, 250 μM ATP with 50 nM of 3′-fluorescein labelled, centrally nicked duplex DNA substrates were incubated with 1 μM Ku and 0.5 μM LigD at 37°C for 15 min, quenching at time points 0, 0.5, 1, 2.5, 5, 7.5, 10, 12.5, 15 min with 98% (v/v) formamide and 200 mM EDTA. The control reaction containing only LigD and DNA was incubated for 15 min. The ligation products (n = 3 technical replicates) were resolved on a 20% denaturing–urea PAGE gel at 200 V for 45 min and were visualized by an Amersham Typhoon Imager (GE Healthcare). Ligation products were quantified using ImageJ (28 (link)). DNA ligation results were plotted as a fraction of DNA product ligated compared to an unligated DNA control. Slopes were calculated by simple linear regression in Prism v.9.0 (GraphPad). Rate was obtained through the calculation of the concentration of product formed over time. p-values were calculated by a two-tailed Welch's t-test in Prism v.9.0 (GraphPad).
9
Dietary Impacts on Zinc Homeostasis
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All statistical analyses were performed using Prism v9.5.1 (GraphPad Software, Inc.). The dietary treatment means ± the standard error of the means (SEM) were plotted for the FluoZin-3 and Zn transporter qPCR assay, and significant differences between treatment groups were determined using a standardized unpaired t-test. Outliers were detected using the ROUT method (Prism v9.5.1) and removed from data analyses when present. This included removal of 1 outlier from the monocyte Fluozin3 analysis (Fig. 2A) and 1 outlier from the CD8 T cell Fluozin3 analysis (Fig. 2D). The statistical analysis was performed using a sample size of 9 steers per dietary treatment, with the experimental unit being individual steers. Initial liver mineral concentrations were analyzed using pre-enrollment status as a covariate but this did not change the outcome and was not used for the analysis in the current manuscript. Plasma trace mineral was analyzed as repeated measures and included treatment × day interactions in the model. Additional details regarding liver and plasma trace mineral status and statistical analyses of dietary treatments are available in Rients et al. (manuscript under review).
10
Quantifying Retained Intron Splicing
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junctionCounts 0.1.0 (https://github.com/ajw2329/junctionCounts ) was first used to extract “retained intron” (RI) alternative splicing events using an annotated genome (gtf) file and the “infer_pairwise_events” command. It was then run against the dup-removed STAR alignments with the “junctionCounts” command against the 344 RI events derived above, using default parameters. The splicing efficiency (% spliced) was calculated by dividing the exon-exon junction count by the sum of the exon-exon and intron-exon count using the equations given in Fig 3A . RI splicing events with less than 100 average read counts between replicates were removed from the total normalized read count, resulting in 287 RI splicing events total (247 single intron RI splicing events and 20 double intron RI splicing events; two intron genes were not further analyzed). The relative splicing efficiency was calculated by normalizing to splicing efficiency values for the appropriate DMSO control. Regression and two-tailed Pearson correlations were calculated using GraphPad Prism v9.4.0. Data was visualized using GraphPad Prism v9.4.0 for macOS.
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