GraphPad Prism (version 8) was used for statistical data analysis. Unpaired t-test or Mann-Whitney non-parametric tests were used to determine the significance in gene expression assays. Survival data was analyzed using Kaplan Meier survival analysis and Gehan-Breslow-Wilcoxin test was used to determine significance. Significance was calculated at p<0.05 and the Grubb’s test on GraphPad QuickCals was used to remove any outliers. A 2-way ANOVA was used to test the effect of vitamin D status or time on DEX plasma levels. Further, Sidak’s multiple comparison test was used to analyze the significance of each effect. For the DEX oral gavage PK study, we used a population modeling approach that is more appropriate for individual animals with variable and sparse repeated serial sampling. We tested sex and VD3 covariates on the parameter CL/F, that is mathematically proportional to AUCinf, as a surrogate for Cmax, these covariates were tested on the volume of distribution parameter V1/F for DEX. Plasma concentration-time (Ct) data in ng/mL for dasatinib were grouped by individual mouse, Vitamin D status, sex, and age, and were analyzed using nonlinear mixed effect (NLME) modeling as implemented in Monolix version 2018R1 (Lixoft SAS, Antony, France). Additionally, Wald test P values were outputted for each covariate effect by the Monolix software.
Quickcal
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QuickCals is a software tool that provides quick and accurate calculation capabilities for various scientific and laboratory applications. It offers a range of mathematical functions and unit conversions to assist researchers and technicians in performing essential calculations efficiently.
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Lab products found in correlation
Spss statistics 20, by IBM (1 mentions)
Prism 5.0a, by GraphPad (1 mentions)
Hygromycin, by Carl Roth (1 mentions)
Lipofectamine ltx and plus reagent, by Thermo Fisher Scientific (1 mentions)
Dmem high glucose glutamax, by Thermo Fisher Scientific (1 mentions)
Tet system approved fetal bovine serum, by Takara Bio (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
21 protocols using quickcal
1
Comprehensive Pharmacokinetic Analysis
2
Statistical Analysis of Experimental Data
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The mean s.d. was calculated using Microsoft Excel. For statistical analysis, an unpaired ‘t’ test was performed to compare the means of two experimental groups using the online software GraphPad Quick Cals, available at http://www.graphpad.com/quickcals/ttest.cfm . The error bars represent s.e.m. [(standard deviation/√n); n=sample size]. Statistical significance is shown with asterisks: *P≤0.05; **P≤0.001; ***P≤0.0001; N.S., not significant. To arrive at the statistically significant sample size for each experiment, we performed power analysis using a previously described model (Cohen, 1988 ), as incorporated in the G*power 3.1 (Faul et al., 2009 (link)) software using the following formula:
where, s.d., standard deviation; Zα/2 and Zβ are type 1 and 2 errors, respectively; d=effect size=difference between mean values. In the worst possible scenarios, we kept the type 1 error to 7% and type 2 error to 80% so that the power was always above 85%.
where, s.d., standard deviation; Zα/2 and Zβ are type 1 and 2 errors, respectively; d=effect size=difference between mean values. In the worst possible scenarios, we kept the type 1 error to 7% and type 2 error to 80% so that the power was always above 85%.
3
Statistical Analysis of Biological Data
4
Genetic Variants and Dengue Vascular Permeability
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The genotype and allele frequencies of each variant were determined. Hardy–Weinberg Equilibrium (HWE) was performed to compute the deviation of the variants tested. Fisher’s exact test was used to determine the significance of the difference in genotype distributions.
Chi-square test with Yates’ correction and/or Fisher’s exact test (when applicable) was performed on the polymorphic variants for association with vascular permeability of dengue using GraphPad QuickCals (http://graphpad.com/quickcalcs/contingency1/ ). Two-sided P values were calculated. Odds ratio (OR) and a Cornfield’s 95% confidence interval (95% CI) were calculated.
Analyses of haplotypes were carried out using HAPLOVIEW [14] (link). Haplotype blocks were constructed according to definition by Gabriel et al. [16] (link). Haplotype blocks and the haplotype frequencies were estimated. Haplotype association tests were performed using chi square test in HAPLOVIEW. Permutation test (1000×) was performed to examine the association significance.
Chi-square test with Yates’ correction and/or Fisher’s exact test (when applicable) was performed on the polymorphic variants for association with vascular permeability of dengue using GraphPad QuickCals (
Analyses of haplotypes were carried out using HAPLOVIEW [14] (link). Haplotype blocks were constructed according to definition by Gabriel et al. [16] (link). Haplotype blocks and the haplotype frequencies were estimated. Haplotype association tests were performed using chi square test in HAPLOVIEW. Permutation test (1000×) was performed to examine the association significance.
5
Statistical Analysis of Experimental Data
6
Diagnostic Accuracy Evaluation of Leishmania Antigens
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The lower limit of positivity (cut-off) for rPeroxidoxin and SLbA was established for optimal sensitivity and specificity using the Receiver Operator Curve (ROC curve). The cut-off was chosen based on the point that provides the maximum of the sum of the sensitivity and specificity [25] (link). The performance of each test was evaluated according to the sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), area under the curve (AUC) and accuracy (AC). The degree of agreement between the ELISA assays using rPeroxidoxin, SLbA or the EIE-LVC kit with a parasitological test (biopsy, aspirate or PCR) was determined by the Kappa index (k) values with 95% confidence intervals and interpreted according to the following Fleiss scale: 0.00–0.20, poor; 0.21–0.40, fair; 0.41–0.60, moderate; 0.61–0.80, good; 0.81–0.99, very good; and 1.00, perfect [26] (link). The one-sample Kolmogorov-Smirnoff test was used to determine whether a variable was normally distributed. For depletion assays, significant differences were detected using unpaired T tests between depleted and undepleted assays. The differences were considered statistically significant at p<0.05. All of the statistical analyses were performed using GraphPad Prism (version 5.0) and GraphPad QuickCals (http://www.graphpad.com/quickcalcs/ ).
7
Analyzing Significance of Experimental Data
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Results were analyzed using a two-tailed Student’s unpaired t test, using GraphPad QuickCals software (unless indicated otherwise) and *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.
8
Antifungal Susceptibility Analysis
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Data were calculated as MIC range, geometric mean MIC (GM MIC), MIC50, and MIC90 in Microsoft Excel version 2019. For geometric means, isolates with MIC’s designated as “equal to or greater than” were given the value as equal to the number. A two-tailed Fisher’s exact test and a two-tailed unpaired t test implemented in GraphPad QuickCals (https://www.graphpad.com/quickcalcs/
9
Zinc Supplementation for ALRI in Children
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After enrolment, the children were randomized to receive zinc supplementation or placebo by an independent person who was not involved in the study. The randomization list was generated by using a computer program (GraphPad QuickCals, La Jolla, CA, USA), in a 1:1 ratio and in a block size of 2. An independent person prepared the randomization schedule and oversaw the packaging and labeling process to ensure the blinding. All investigators, children, parents, and guardians were blinded throughout the study. The randomization codes were opened after the completion of the study.
The children in the treatment group received chelated zinc as zinc bis-glycinate (15 mg elemental zinc) twice a day until their discharge from the hospital, or up to a maximum of 7 days. Children in the control group received an ORS-based placebo, with similar appearance and taste to zinc powder. Both the zinc and placebo were manufactured and supplied by Qualimed (Bangkok, Thailand) as a powder in an identical single- dose sachet. The contents of the sachet were dissolved in a glass of water before administration. The treatment of ALRI, as well as the observation and the discharge decisions, were made by the attending physicians, who were not involved in the implementation phase of the study.
The children in the treatment group received chelated zinc as zinc bis-glycinate (15 mg elemental zinc) twice a day until their discharge from the hospital, or up to a maximum of 7 days. Children in the control group received an ORS-based placebo, with similar appearance and taste to zinc powder. Both the zinc and placebo were manufactured and supplied by Qualimed (Bangkok, Thailand) as a powder in an identical single- dose sachet. The contents of the sachet were dissolved in a glass of water before administration. The treatment of ALRI, as well as the observation and the discharge decisions, were made by the attending physicians, who were not involved in the implementation phase of the study.
10
Upper Canine Color Difference Analysis
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The sample size was calculated based on the upper canines’ color difference (ΔE00 — a primary outcome) recorded in a previously performed pilot study with 30 randomized participants (GraphPad QuickCals, http://www.graphpad.com/quickcalcs/randomize1.cfm ), using the G*Power 3.1 software (Heinrich-Heine-Universität, Düsseldorf, Germany). The size effect was calculated based on the perceptibility threshold ΔE00 = 0.8 with a standard deviation of 0.81 [57 (link), 58 (link)]. Considering an F test (one-way ANOVA) with a significance level of 5% and a power of 80%, a minimum of 20 participants per group was required. To offset a possible attrition bias, 50% was added to each group, resulting in 30 patients’ samples (a total of 90 patients).
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