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

NuRD-mediated deacetylation of H3K27 facilitates recruitment of Polycomb Repressive Complex 2 to direct gene repression

The NURD and Polycomb complexes PRC1 and PRC2 have been implicated in stem cell differentiation although their molecular roles are unclear. This study identifies a common set of genes that are sequentially regulated by these complexes, the NURD complex deacetylates H3K27 as a prerequisite for subsequent methylation by PRC2

Dedifferentiation and Proliferation of Mammalian Cardiomyocytes

It has long been thought that mammalian cardiomyocytes are terminally-differentiated and unable to proliferate. However, myocytes in more primitive animals such as zebrafish are able to dedifferentiate and proliferate to regenerate amputated cardiac muscle.Here we test the hypothesis that mature mammalian cardiomyocytes retain substantial cellular plasticity, including the ability to dedifferentiate, proliferate, and acquire progenitor cell phenotypes. Two complementary methods were used: 1) cardiomyocyte purification from rat hearts, and 2) genetic fate mapping in cardiac explants from bi-transgenic mice. Cardiomyocytes isolated from rodent hearts were purified by multiple centrifugation and Percoll gradient separation steps, and the purity verified by immunostaining and RT-PCR. Within days in culture, purified cardiomyocytes lost their characteristic electrophysiological properties and striations, flattened and began to divide, as confirmed by proliferation markers and BrdU incorporation. Many dedifferentiated cardiomyocytes went on to express the stem cell antigen c-kit, and the early cardiac transcription factors GATA4 and Nkx2.5. Underlying these changes, inhibitory cell cycle molecules were suppressed in myocyte-derived cells (MDCs), while microRNAs known to orchestrate proliferation and pluripotency increased dramatically. Some, but not all, MDCs self-organized into spheres and re-differentiated into myocytes and endothelial cells in vitro. Cell fate tracking of cardiomyocytes from 4-OH-Tamoxifen-treated double-transgenic MerCreMer/ZEG mouse hearts revealed that green fluorescent protein (GFP) continues to be expressed in dedifferentiated cardiomyocytes, two-thirds of which were also c-kit(+).Contradicting the prevailing view that they are terminally-differentiated, postnatal mammalian cardiomyocytes are instead capable of substantial plasticity. Dedifferentiation of myocytes facilitates proliferation and confers a degree of stemness, including the expression of c-kit and the capacity for multipotency

Telomerase variants in myelodysplastic syndromes

BACKGROUND: Alternative splicing of hTERT mRNA contributes to the regulation of telomerase activity in normal and neoplastic cells. The deleted variant A (Adel), lacking 36nts in the beginning of exon 6, blocks telomerase activity while the B deleted variant (Bdel), lacking exons 7 and 8, exhibits no action in terms of telomerase activation. The expression of hTERT mRNA has been investigated in myeloid malignancies with controversial results. The expression of hTERT mRNA variants has not been explored in MDS patients. AIM: To investigate the expression of hTERT mRNA variant transcripts A+B+ (contained in the full-length product), Adel and Bdel in the bone marrow of MDS and AML untreated patients. METHODS: Bone marrow aspirates from 27 patients with refractory anaemia or refractory cytopenia with multilineage dysplasia (RA or RCMD, defined as group A), 13 patients with refractory anaemia with excess of blasts (RAEB1 or RAEB2, defined as group B) and 17 AML patients (defined as group C) were studied. Relative expression of the above transcripts was assessed on total RNA with reverse transcription followed by real-time polymerase chain reaction (PCR). The data obtained were analyzed by using Pearson chi-square and Kruskal Wallis statistics. All individuals signed informed consent before sampling. RESULTS: hTERT mRNA was detected in 10, 4 and 3 patients of groups A, B and C respectively. The full length product (A+B+) was detected in groups A, B and C (2/27, 4/13, 3/17 respectively) (pA/B=0.053). Adel isoform was detected in groups A (7/27) and B (2/13) but not in group C (0/17) (pA/C=0.022). Bdel isoform was observed in group A (3/27), B (3/13) and C (2/17). Co-expression of isoforms was observed in 2 patients of A group (Adel /Bdel, Adel/A+B+), 4 patients of group B (1 Adel/A+B+, 1 Bdel/A+B+, 2 Adel/Bdel/A+B+) and two patients of group C (Bdel/A+B+). Relative expression levels of Adel, Bdel, and A+B+ transcripts were not significantly different among patient groups. CONCLUSIONS: Alternatively spliced hTERT variants either lack a critical reverse-transcriptase motif or produce a non-functional reverse-transcriptase. Therefore, cells may control telomerase activity by switching their hTERT mRNA variant expression profile. Our results indicate that aberrations in hTERT variant profiles, especially regarding the Adel isoform, may be implicated in the pathogenesis and progression of MDS. The increased incidence of Adel positivity among RA or RCMD patients may be connected with a decline in telomerase activity and therefore contribute to the establishment of chromosomal abnormalities. This observation underlines the need to distinguish between the different hTERT mRNA variants when investigating hTERT expression in MDS patients.Τα μυελοδυσπλαστικά σύνδρομα (ΜΔΣ) αποτελούν μία ετερογενή ομάδα κλωνικών διαταραχών του αρχέγονου αιμοποιητικού κυττάρου. Τελομερή ονομάζονται τα άκρα των ευκαρυωτικών χρωμοσωμάτων που αποτελούνται από επαναλαμβανόμενες αλληλουχίες βάσεων και προσδίδουν γενωμική σταθερότητα στα κύτταρα. Το μήκος των τελομερών συνδέθηκε τόσο με την ύπαρξη κυτταροπενιών όσο και με την μετατροπή προς ΟΜΛ με αποτέλεσμα να θεωρηθεί ως ένας πιθανός συμπληρωματικός προγνωστικός δείκτης για τους ασθενείς με MΔΣ. Η τελομεράση είναι ένα ριβονουκλεϊκοπρωτεϊνικό ένζυμο που προσθέτει TTAGGG τμήματα στα τελομερή. Η τελομεράση αποτελείται από δύο κύριες υπομονάδες, τη δομική RNA υπομονάδα (human Telomerase RNA ComponenthTERC) και την καταλυτική υπομονάδα (human Telomerase Reverse Transcrpriptase-hTERT). Η υπομονάδα hTERC λειτουργεί ως μήτρα για την προσθήκη των νουκλεοτιδίων στα τελομερή ενώ η υπομονάδα hTERT παρουσιάζει δράση ανάστροφης μεταγραφάσης και υφίσταται μετα-μεταγραφική μετατροπή με αποτέλεσμα να προκύπτουν τρεις κύριες ισομορφές: η ισομορφή Αdel που δρα ανασταλτικά στην ενεργοποίηση της τελομεράσης, η Bdel που είναι ανενεργής σε σχέση με την ενεργοποίηση του ενζύμου και η πλήρης ισομορφή (Α+Β) που είναι η μόνη που δρα καταλυτικά για τη λειτουργία της τελομεράσης. Σκοπός της παρούσας μελέτης ήταν η μελέτη της έκφρασης σε επίπεδο mRNA των ισομορφών hTERT καθώς και των p73, ΟCT4 και OCT4A. Μελετήθηκαν 40 ασθενείς με ΜΔΣ: 27 ασθενείς με 5% βλάστες εμφανίζεται συχνότερα η ενεργή ισομορφή (Α+Β) που ασκεί καταλυτική δράση στη λειτουργία της δομικής υπομονάδας της τελομεράσης. Συμπερασματικά η τελομεράση ενδέχεται να συμμετέχει στην παθογένεια αλλά και στην εξέλιξη των ΜΔΣ προς ΟΜΛ με μετατροπή των ισομορφών hTERT: η ισομορφή Adel που ευνοεί τη συσσώρευση γενετικών βλαβών και δρα αποπτωτικά αντικαθίσταται στα υψηλότερου κινδύνου ΜΔΣ από την ισομορφή (A+B) που δρα αντιαποπτωτικά

Protocol for unbiased, consolidated variant calling from whole exome sequencing data.

Whole Exome Sequencing (WES) is used for querying DNA variants using the protein coding parts of genomes (exomes). However, WES analysis can be challenging because of the complexity of the data. Here, we describe a consolidated protocol for unbiased WES analysis. The protocol uses three variant callers (HaplotypeCaller, FreeBayes, and DeepVariant), which have different underlying models. We provide detailed execution steps, as well as basic variant filtering, annotation, visualization, and consolidation aspects

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

The role of A-to-I RNA editing in infections by RNA viruses: Possible implications for SARS-CoV-2 infection

RNA editing is a fundamental biological process with 2 major forms, namely adenosine-to-inosine (A-to-I, recognized as A-to-G) and cytosine-to-uracil (C-to-U) deamination, mediated by ADAR and APOBEC enzyme families, respectively. A-to-I RNA editing has been shown to directly affect the genome/transcriptome of RNA viruses with significant repercussions for viral protein synthesis, proliferation and infectivity, while it also affects recognition of double-stranded RNAs by cytosolic receptors controlling the host innate immune response. Recent evidence suggests that RNA editing may be present in SARS-CoV-2 genome/transcriptome. The majority of mapped mutations in SARS-CoV-2 genome are A-to-G/U-to-C(opposite strand) and C-to-U/G-to-A(opposite strand) substitutions comprising potential ADAR-/APOBEC-mediated deamination events. A single nucleotide substitution can have dramatic effects on SARS-CoV-2 infectivity as shown by the D614G(A-to-G) substitution in the spike protein. Future studies utilizing serial sampling from patients with COVID-19 are warranted to delineate whether RNA editing affects viral replication and/or the host immune response to SARS-CoV-2. © 2021 Elsevier Inc

A Meta-Epidemiological Study of Positive Results in Clinical Nutrition Research: The Good, the Bad and the Ugly of Statistically Significant Findings

Positive (statistically significant) findings are easily produced in nutrition research when specific aspects of the research design and analysis are not accounted for. To address this issue, recently, a pledge was made to reform nutrition research and improve scientific trust on the science, encompass research transparency and achieve reproducibility. The aim of the present meta-epidemiological study was to evaluate the statistical significance status of research items published in three academic journals, all with a focus on clinical nutrition science and assessing certain methodological/transparency issues. All research items were published between the years 2015 and 2019. Study design, primary and secondary findings, sample size and age group, funding sources, positivist findings, the existence of a published research protocol and the adjustment of nutrients/dietary indexes to the energy intake (EI) of participants, were extracted for each study. Out of 2127 studies in total, those with positive findings consisted of the majority, in all three journals. Most studies had a published research protocol, however, this was mainly due to the randomized controlled trials and not to the evidence-synthesis studies. No differences were found in the distribution of positive findings according to the existence/inexistence of a published research protocol. In the pooled sample of studies, positive findings differed according to study design and more significant findings were reported by researchers failing to report any funding source. The majority of items published in the three journals (65.9%) failed to account for the EI of participants. The present results indicate that there is still room for the improvement of nutrition research in terms of design, analyses and reporting. © 2022 by the authors