Timely and accurate detection of the causal agent of a disease is crucial to restrict suffering and save lives. Mere symptoms are often not enough to detect the root cause of the disease. Better diagnostics applied for screening at a population level and sensitive detection assays remain the crucial component of disease surveillance which may include clinical, plant, and environmental samples, including wastewater. The recent advances in genome sequencing, nucleic acid amplification, and detection methods have revolutionized nucleic acid-based testing (NABing) and screening assays. A typical NABing assay consists of three modules: isolation of the nucleic acid from the collected sample, identification of the target sequence, and final reading the target with the help of a signal, which may be in the form of color, fluorescence, etc. Here, we review current NABing assays covering the different aspects of all three modules. We also describe the frequently used target amplification or signal amplification procedures along with the variety of applications of this fast-evolving technology and challenges in implementation of NABing in the context of disease management especially in low-resource settings.

Human mesenchymal stromal/stem cells (hMSCs) emerged as a promising therapeutic tool for ischemic disorders, due to their ability to regenerate damaged tissues, promote angiogenesis and reduce inflammation, leading to encouraging, but still limited results. The outcomes in clinical trials exploring hMSC therapy are influenced by low cell retention and survival in affected tissues, partially influenced by lesion's microenvironment, where low oxygen conditions (i.e. hypoxia) and inflammation coexist. Hypoxia and inflammation are pathophysiological stresses, sharing common activators, such as hypoxia-inducible factors (HIFs) and NF-κB. HIF1α and HIF2α respond essentially to hypoxia, activating pathways involved in tissue repair. Little is known about the regulation of HIF3α. Here we investigated the role of HIF3α in vitro and in vivo. Human MSCs expressed HIF3α, differentially regulated by pro-inflammatory cytokines in an oxygen-independent manner, a novel and still uncharacterized mechanism, where NF-κB is critical for its expression. We investigated if epigenetic modifications are involved in HIF3α expression by methylation-specific PCR and histone modifications. Robust hypermethylation of histone H3 was observed across HIF3A locus driven by pro-inflammatory cytokines. Experiments in a murine model of arteriotomy highlighted the activation of Hif3α expression in infiltrated inflammatory cells, suggesting a new role for Hif3α in inflammation in vivo.

Methylation of DNA, the most experimentally accessible epigenetic alteration of eukaryotic cells, has generated an extensive literature and an abundance of analytical tools. The term "methylome" (referring to the complete set of cytosine modifications in a genome) is appearing with greater frequency in the literature, reflecting the growing number of researchers in the field. Here we introduce a set of robust protocols for methods that can be performed routinely for the elucidation of DNA chemical modifications involving methylation of cytosine. The strengths and limitations of each approach are also discussed.