Hypothesis testing for two population means: parametric or non-parametric test?

The parametric Welch $t$-test and the non-parametric Wilcoxon-Mann-Whitney test are the most commonly used two independent sample means tests. More recent testing approaches include the non-parametric, empirical likelihood and exponential empirical likelihood. However, the applicability of these non-parametric likelihood testing procedures is limited partially because of their tendency to inflate the type I error in small sized samples. In order to circumvent the type I error problem, we propose simple calibrations using the $t$ distribution and bootstrapping. The two non-parametric likelihood testing procedures, with and without those calibrations, are then compared against the Wilcoxon-Mann-Whitney test and the Welch $t$-test. The comparisons are implemented via extensive Monte Carlo simulations on the grounds of type I error and power in small/medium sized samples generated from various non-normal populations. The simulation studies clearly demonstrate that a) the $t$ calibration improves the type I error of the empirical likelihood, b) bootstrap calibration improves the type I error of both non-parametric likelihoods, c) the Welch $t$-test with or without bootstrap calibration attains the type I error and produces similar levels of power with the former testing procedures, and d) the Wilcoxon-Mann-Whitney test produces inflated type I error while the computation of an exact p-value is not feasible in the presence of ties with discrete data. Further, an application to real gene expression data illustrates the computational high cost and thus the impracticality of the non parametric likelihoods. Overall, the Welch t-test, which is highly computationally efficient and readily interpretable, is shown to be the best method when testing equality of two population means.Comment: Accepted for publication in the Journal of Statistical Computation and Simulatio

An in silico method for studying the phosphorylation in association to active sites

Post-translational modifications (PTMs) occur to a vast amount of proteins and the most common post-translational modification (PTM) is phosphorylation. Phosphorylation and dephosphorylation, regulate protein functionality by turning the protein active sites (sites with important biological function) on and off. Therefore, identification of a protein’s phosphorylated residues and determination of their role are of paramount importance, especially for proteins driving diseases. Notwithstanding the multiple methodologies for identifying phosphorylated residues, literature lacks of methodologies for determination of their role. For this reason, we created a method that aims to enhance the understanding of a protein’s regulation by phosphorylation as well as to aid the design of more directed and lower-cost experiments. Our method uses the PhosphoKin tool, which predicts new phosphorylated residues in a given protein sequence, identifies the possibly responsible kinases for the protein’s experimentally observed phosphorylated residues and links all phosphorylated residues as well as their kinases with the protein’s active sites. Our method assesses the impact of the examined kinases in the protein’s phosphorylation and is suitable for associations between specific group of kinases and active sites. Also, it suggests the illustration of a phosphorylation map of the protein that is useful for further analysis

Hypothesis testing for two population means: parametric or non-parametric test?

The parametric Welch t-test and the non-parametric Wilcoxon–Mann–Whitney, empirical and exponential empirical likelihood tests are commonly used for hypothesis testing of two population means. In order to circumvent the inflated type I error problem of the nonparametric likelihood testing procedures, a simple calibration using the t distribution and bootstrapping is proposed. Those testing procedures are then being compared via extensive Monte Carlo simulations on the grounds of type I error and power. Evidence is provided supporting that (a) the t calibration and bootstrap improve the type I error of the non-parametric likelihoods, (b) the Welch ttest attains the type I error and produces high levels of power, and (c) the Wilcoxon–Mann–Whitney test produces inflated type I error while computation of the exact p-value is not feasible in the presence of ties. An application to real gene expression data illustrates the computational superiority of the Welch t-test

SARS-CoV-2 mRNA Dual Immunization Induces Innate Transcriptional Signatures, Establishes T-Cell Memory and Coordinates the Recall Response

Background: mRNA vaccines have played a crucial role in controlling the SARS-CoV-2 global pandemic. However, the immunological mechanisms involved in the induction, magnitude and longevity of mRNA-vaccine-induced protective immunity are still unclear. Methods: In our study, we used whole-RNA sequencing along with detailed immunophenotyping of antigen-specific T cells and humoral RBD-specific response to dual immunization with the Pfizer–BioNTech mRNA vaccine (BNT162b2) and correlated them with response to an additional dose, administered 10 months later, in order to comprehensively profile the immune response of healthy volunteers to BNT162b2. Results: Primary dual immunization induced upregulation of the Type I interferon pathway and generated spike protein (S)-specific IFN-γ+ and TNF-α+ CD4 T cells, S-specific memory CD4 T cells, and RBD-specific antibodies against SARS-CoV-2. S-specific CD4 T cells induced by the primary series correlated with the RBD-specific antibody titers to a third dose. Conclusions: This study demonstrates the induction of both innate and adaptive immunity in response to the BNT162b2 mRNA vaccine in a coordinated manner and identifies the central role of primarily induced CD4+ T cells as a predictive biomarker of the magnitude of anamnestic immune response

Distinct innate and adaptive immunity phenotypic profile at the circulating single-cell level in psoriatic arthritis

Mass cytometry was employed to investigate 47 circulating leukocyte subsets in patients with active psoriatic arthritis (PsA, n = 16) compared to healthy controls (n = 13), seropositive (RF and/or anti-CCP, n = 12) and seronegative (n = 9) RA patients. Comparing PsA to controls, different cell frequencies were found in both innate and adaptive immunity cell subsets, as well as in cells bridging innate and adaptive immunity. In some T cell subsets increased costimulatory molecules' expression in PsA, was also noted.No changes were observed in patients who remained disease-active after 3 months of treatment, in contrast to those who achieved remission/low-disease activity. Comparing PsA to seropositive RA, elevated frequencies of naïve and activated CD8+ T cells, B cells, MAIT/iNKT and ILCs were found, while the opposite was the case for terminal effector, senescent, and Th2-like cells. Strikingly, the composition of the leukocyte pool in PsA was comparable to seronegative RA, providing evidence for the pathogenetic similarities between these two entities

Blood transcriptomes of anti-SARS-CoV2 antibody positive healthy individuals with prior asymptomatic versus clinical infection

The reasons behind the clinical variability of SARS-CoV-2 infection, ranging from asymptomatic infection to lethal disease, are still unclear. We performed genome-wide transcriptional whole-blood RNA sequencing, bioinformatics analysis and PCR validation to test the hypothesis that immune response-related gene signatures reflecting baseline may differ between healthy individuals, with an equally robust antibody response, who experienced an entirely asymptomatic (n=17) versus clinical SARS-CoV-2 infection (n=15) in the past months (mean of 14 weeks). Among 12.789 protein-coding genes analysed, we identified six and nine genes with significantly decreased or increased expression, respectively, in those with prior asymptomatic infection relatively to those with clinical infection. All six genes with decreased expression (IFIT3, IFI44L, RSAD2, FOLR3, PI3, ALOX15), are involved in innate immune response while the first two are interferon-induced proteins. Among genes with increased expression six are involved in immune response (GZMH, CLEC1B, CLEC12A), viral mRNA translation (GCAT), energy metabolism (CACNA2D2) and oxidative stress response (ENC1). Notably, 8/15 differentially expressed genes are regulated by interferons. Our results suggest that subtle differences at baseline expression of innate immunity-related genes may be associated with an asymptomatic disease course in SARS-CoV-2 infection. Whether a certain gene signature predicts, or not, those who will develop a more efficient immune response upon exposure to SARS-CoV-2, with implications for prioritization for vaccination, warrant further study. © Copyright © 2021 Sfikakis, Verrou, Ampatziadis-Michailidis, Tsitsilonis, Paraskevis, Kastritis, Lianidou, Moutsatsou, Terpos, Trougakos, Chini, Manoloukos, Moulos, Pavlopoulos, Kollias, Hatzis and Dimopoulos