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As the COVID-19 pandemic winds down, it leaves behind the serious concern that future, even more disruptive pandemics may eventually surface. One of the crucial steps in handling the SARS-CoV-2 pandemic was being able to detect the presence of the virus in an accurate and timely manner, to then develop policies counteracting the spread. Nevertheless, as the pandemic evolved, new variants with potentially dangerous mutations appeared. Faced by these developments, it becomes clear that there is a need for fast and reliable techniques to create highly specific molecular tests, able to uniquely identify VOCs. Using an automated pipeline built around evolutionary algorithms, we designed primer sets for SARS-CoV-2 (main lineage) and for VOC, B.1.1.7 (Alpha) and B.1.1.529 (Omicron). Starting from sequences openly available in the GISAID repository, our pipeline was able to deliver the primer sets for the main lineage and each variant in a matter of hours. Preliminary in-silico validation showed that the sequences in the primer sets featured high accuracy. A pilot test in a laboratory setting confirmed the results: the developed primers were favorably compared against existing commercial versions for the main lineage, and the specific versions for the VOCs B.1.1.7 and B.1.1.529 were clinically tested successfully., (© 2023. Springer Nature Limited.)

Background: Type 2 diabetes (T2D) is associated with coronary microvascular dysfunction, which is thought to contribute to compromised diastolic function, ultimately culminating in heart failure with preserved ejection fraction (HFpEF). The molecular mechanisms remain incompletely understood, and no early diagnostics are available. We sought to gain insight into biomarkers and potential mechanisms of microvascular dysfunction in obese mouse (db/db) and lean rat (Goto-Kakizaki) pre-clinical models of T2D-associated diastolic dysfunction., Methods: The microRNA (miRNA) content of circulating extracellular vesicles (EVs) was assessed in T2D models to identify biomarkers of coronary microvascular dysfunction/rarefaction. The potential source of circulating EV-encapsulated miRNAs was determined, and the mechanisms of induction and the function of candidate miRNAs were assessed in endothelial cells (ECs)., Results: We found an increase in miR-30d-5p and miR-30e-5p in circulating EVs that coincided with indices of coronary microvascular EC dysfunction (i.e., markers of oxidative stress, DNA damage/senescence) and rarefaction, and preceded echocardiographic evidence of diastolic dysfunction. These miRNAs may serve as biomarkers of coronary microvascular dysfunction as they are upregulated in ECs of the left ventricle of the heart, but not other organs, in db/db mice. Furthermore, the miR-30 family is secreted in EVs from senescent ECs in culture, and ECs with senescent-like characteristics are present in the db/db heart. Assessment of miR-30 target pathways revealed a network of genes involved in fatty acid biosynthesis and metabolism. Over-expression of miR-30e in cultured ECs increased fatty acid β-oxidation and the production of reactive oxygen species and lipid peroxidation, while inhibiting the miR-30 family decreased fatty acid β-oxidation. Additionally, miR-30e over-expression synergized with fatty acid exposure to down-regulate the expression of eNOS, a key regulator of microvascular and cardiomyocyte function. Finally, knock-down of the miR-30 family in db/db mice decreased markers of oxidative stress and DNA damage/senescence in the microvascular endothelium., Conclusions: MiR-30d/e represent early biomarkers and potential therapeutic targets that are indicative of the development of diastolic dysfunction and may reflect altered EC fatty acid metabolism and microvascular dysfunction in the diabetic heart., (© 2022. The Author(s).)

In this paper, deep learning is coupled with explainable artificial intelligence techniques for the discovery of representative genomic sequences in SARS-CoV-2. A convolutional neural network classifier is first trained on 553 sequences from the National Genomics Data Center repository, separating the genome of different virus strains from the Coronavirus family with 98.73% accuracy. The network's behavior is then analyzed, to discover sequences used by the model to identify SARS-CoV-2, ultimately uncovering sequences exclusive to it. The discovered sequences are validated on samples from the National Center for Biotechnology Information and Global Initiative on Sharing All Influenza Data repositories, and are proven to be able to separate SARS-CoV-2 from different virus strains with near-perfect accuracy. Next, one of the sequences is selected to generate a primer set, and tested against other state-of-the-art primer sets, obtaining competitive results. Finally, the primer is synthesized and tested on patient samples (n = 6 previously tested positive), delivering a sensitivity similar to routine diagnostic methods, and 100% specificity. The proposed methodology has a substantial added value over existing methods, as it is able to both automatically identify promising primer sets for a virus from a limited amount of data, and deliver effective results in a minimal amount of time. Considering the possibility of future pandemics, these characteristics are invaluable to promptly create specific detection methods for diagnostics.

IAMBEE is a web server designed for the Identification of Adaptive Mutations in Bacterial Evolution Experiments (IAMBEE). Input data consist of genotype information obtained from independently evolved clonal populations or strains that show the same adapted behavior (phenotype). To distinguish adaptive from passenger mutations, IAMBEE searches for neighborhoods in an organism-specific interaction network that are recurrently mutated in the adapted populations. This search for recurrently mutated network neighborhoods, as proxies for pathways is driven by additional information on the functional impact of the observed genetic changes and their dynamics during adaptive evolution. In addition, the search explicitly accounts for the differences in mutation rate between the independently evolved populations. Using this approach, IAMBEE allows exploiting parallel evolution to identify adaptive pathways. The web-server is freely available at http://bioinformatics.intec.ugent.be/iambee/ with no login requirement., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of the establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the major Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to distinguish subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on the cooperativity between the 6 known Bicoid binding sites in the hb promoter region, assuming rate limiting concentrations of the Bicoid transcription factor at the boundary, is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that a simple model based only on the cooperative binding of Bicoid is not sufficient to describe the spatiotemporal dynamics of early hb expression., Competing Interests: The authors have declared that no competing interests exist.

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-off between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the gene's ability to read positional information along the embryo's anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies., Competing Interests: The authors have declared that no competing interests exist.

Telomeres are maintained in a heterochromatic state that represses transcription of subtelomeric genes, a phenomenon known as telomere position effect. Nevertheless, telomeric DNA is actively transcribed, leading to the synthesis of telomeric repeat-containing noncoding RNA or TERRA. This nuclear noncoding RNA has been proposed to play important roles at telomeres, regulating their silencing, capping, repair and elongation by telomerase. In the budding yeast Saccharomyces cerevisiae, TERRA accumulation is repressed by telomeric silencing and the Rat1 exonuclease. On the other hand, telomere shortening promotes expression of TERRA. So far, little is known about the biological processes that induce TERRA expression in yeast. Understanding the dynamics of TERRA expression and localization is essential to define its function in telomere biology. Here, we aim to study the dynamics of TERRA expression during yeast cell growth. Using live-cell imaging, RNA-FISH and quantitative RT-PCR, we show that TERRA expression is induced as yeast cells undergo diauxic shift, a lag phase during which yeast cells switch their metabolism from anaerobic fermentation to oxidative respiration. This induction is transient as TERRA levels decrease during post-diauxic shift. The increased expression of TERRA is not due to the shortening of telomeres or increased stability of this transcript. Surprisingly, this induction is coincident with a cytoplasmic accumulation of TERRA molecules. Our results suggest that TERRA transcripts may play extranuclear functions with important implications in telomere biology and add a novel layer of complexity in the interplay between telomere biology, metabolism and stress response.

We present the LiveFly toolbox for quantitative analysis of transcription dynamics in live Drosophila embryos. The toolbox allows users to process two-color 3D confocal movies acquired using nuclei-labeling and the fluorescent RNA-tagging system described in the previous chapter and export the nuclei's position as a function of time, their lineages and the intensity traces of the active loci. The toolbox, which is tailored for the context of Drosophila early development, is semiautomatic, and requires minimal user intervention. It also includes a tool to combine data from multiple movies and visualize several features of the intensity traces and the expression pattern.

Live imaging has been used in recent years for the understanding of dynamic processes in biology, such as embryo development. This was made possible by a combination of advancements in microscopy, leading to improved signal-to-noise ratios and better spatial and temporal resolutions, and by the development of new fluorescence markers, allowing for the quantification of protein expression and transcriptional dynamics in vivo. Here we describe a general protocol, which can be used in standard confocal microscopes to image early Drosophila melanogaster embryos, in order to learn about the transcriptional dynamics of a fluorescently labeled RNA.

Common variable immunodeficiency (CVID) is a syndrome with predominantly defective B cell function. However, abnormalities in the number and function of other lymphocyte subpopulations in peripheral blood (PB) have been described in most patients. We have analysed the distribution of iNKT cell subpopulations in the PB of CVID patients and the ability of these cells to provide in vitro cognate B cell help. The total of iNKT cells was reduced in the PB of CVID patients, especially CD4+, CD4-/CD8- and CCR5+/CXCR3+. These findings were associated with an enrichment of memory-like and a tendency towards a reduction in TNF-α-expressing effector iNKT cells in the peripheral blood mononuclear cells (PBMC) of CVID patients. Moreover, an accumulation of follicular helper iNKT cells in the PB of CVID patients was demonstrated. CVID αGalCer-pulsed iNKT cells are not able to induce autologous B cell proliferation although they do induce proliferation to healthy donor B cells. Interestingly, autologous and heterologous co-cultures did not differ in the amount of immunoglobulin secreted by B cells in vitro. Finally, reduced intracellular SAP expression in iNKT cells and other lymphocytes in the blood from CVID patients was observed. These results provide further insights into the immunological mechanisms underlying the iNKT cell defects and the potential targets to improve B cell help in CVID., (© 2017 The Foundation for the Scandinavian Journal of Immunology.)