SOX2 and OCT4 mRNA-Expressing Cells, Detected by Molecular Beacons, Localize to the Center of Neurospheres during Differentiation

Neurospheres are used as in vitro assay to measure the properties of neural stem cells. To investigate the molecular and phenotypic heterogeneity of neurospheres, molecular beacons (MBs) targeted against the stem cell markers OCT4 and SOX2 were designed, and synthesized with a 2’-O-methyl RNA backbone. OCT4 and SOX2 MBs were transfected into human embryonic mesencephalon derived cells, which spontaneously form neurospheres when grown on poly-L-ornitine/fibronectin matrix and medium complemented with bFGF. OCT4 and SOX2 gene expression were tracked in individual cell using the MBs. Quantitative image analysis every day for seven days showed that the OCT4 and SOX2 mRNA-expressing cells clustered in the centre of the neurospheres cultured in differentiation medium. By contrast, cells at the periphery of the differentiating spheres developed neurite outgrowths and expressed the tyrosine hydroxylase protein, indicating terminal differentiation. Neurospheres cultured in growth medium contained OCT4 and SOX2-positive cells distributed throughout the entire sphere, and no differentiating neurones. Gene expression of SOX2 and OCT4 mRNA detected by MBs correlated well with gene and protein expression measured by qRT-PCR and immunostaining, respectively. These experimental data support the theoretical model that stem cells cluster in the centre of neurospheres, and demonstrate the use of MBs for the spatial localization of specific gene-expressing cells within heterogeneous cell populations

Long Non-Coding RNAs in Cardiac and Pulmonary Fibroblasts and Fibrosis

The cardiopulmonary system delivers oxygen throughout the body via blood circulation. It is an essential part of the body to sustain the lives of organisms. The integral parts of the cardiopulmonary system—the heart and lungs—are constantly exposed to damaging agents (e.g., dust, viruses), and can be greatly affected by injuries caused by dysfunction in tissues (e.g., myocardial infarction). When damaged, mesenchymal cells, such as fibroblasts, are activated to become myofibroblasts to initiate fibrosis as part of a regenerative mechanism. In diseased states, the excess accumulation of extracellular matrices secreted by myofibroblasts results in further dysfunction in the damaged organs. These fibrotic tissues cannot easily be removed. Thus, there is a growing interest in understanding the fibrotic process, as well as finding biomolecules that can be targets for slowing down or potentially stopping fibrosis. Among these biomolecules, the interest in studying long non-coding RNAs (lncRNAs; any non-protein-coding RNAs longer than 200 nucleotides) has intensified in recent years. In this commentary, we summarize the current status of lncRNA research in the cardiopulmonary system by focusing on cardiac and pulmonary fibrosis

Functional roles of epitranscriptomic marks in the cardiovascular system and disease: a narrative review

BACKGROUND AND OBJECTIVE: The recent emergence of epitranscriptomics provides an avenue for identifying RNA modifications implicated in the pathophysiology of human disease. To date, over 170 RNA modifications have been identified; these modifications are important because they can affect the fate of RNAs, including their decay, maturation, splicing, stability, and translational efficiency. Although RNA modifications have been reported in many tissues and disease contexts, detailed functional studies in the heart and cardiovascular disease are only beginning to be reported. METHODS: The search for relevant articles related to epitranscriptomics was conducted by focusing on the cardiovascular system and disease in the PubMed database. KEY CONTENT AND FINDINGS: We summarize the recent findings of three epitranscriptomic marks—N6-methyladenosine (m(6)A), adenosine to inosine (A-to-I) RNA editing, and 5-methylcytosine (m(5)C) as other epitranscriptomic marks are not studied extensively in the cardiovascular system and disease. CONCLUSIONS: In this narrative review, the current status of cardiac epitranscriptomics is summarized to raise the awareness of this important field of study

Artemin and an Artemin-Derived Peptide, Artefin, Induce Neuronal Survival, and Differentiation Through Ret and NCAM

Artemin (ARTN) is a neurotrophic factor from the GDNF family ligands (GFLs) that is involved in development of the nervous system and neuronal differentiation and survival. ARTN signals through a complex receptor system consisting of the RET receptor tyrosine kinase and a glycosylphosphatidylinositol-anchored co-receptor GFL receptor α, GFRα3. We found that ARTN binds directly to neural cell adhesion molecule (NCAM) and that ARTN-induced neuritogenesis requires NCAM expression and activation of NCAM-associated signaling partners, thus corroborating that NCAM is an alternative receptor for ARTN. We designed a small peptide, artefin, that could interact with GFRα3 and demonstrated that this peptide agonist induces RET phosphorylation and mimics the biological functions of ARTN – neuroprotection and neurite outgrowth. Moreover, artefin mimicked the binding of ARTN to NCAM and required NCAM expression and activation for its neurite elongation effect, thereby suggesting that artefin represents a binding site for NCAM within ARTN. We showed that biological effects of ARTN and artefin can be inhibited by abrogation of both NCAM and RET, suggesting a more complex signaling mechanism that previously thought. As NCAM plays a significant role in neurodevelopment, regeneration, and synaptic plasticity we suggest that ARTN and its mimetics are promising candidates for treatment of neurological disorders and warrant further investigations

The current status of gene expression profilings in COVID-19 patients

BACKGROUND: The global pandemic of coronavirus disease 2019 (COVID‐19) caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has swept through every part of the world. Because of its impact, international efforts have been underway to identify the variants of SARS‐CoV‐2 by genome sequencing and to understand the gene expression changes in COVID‐19 patients compared to healthy donors using RNA sequencing (RNA‐seq) assay. Within the last two and half years since the emergence of SARS‐CoV‐2, a large number of OMICS data of COVID‐19 patients have accumulated. Yet, we are still far from understanding the disease mechanism. Further, many people suffer from long‐term effects of COVID‐19; calling for a more systematic way to data mine the generated OMICS data, especially RNA‐seq data. METHODS: By searching gene expression omnibus (GEO) using the key terms, COVID‐19 and RNA‐seq, 108 GEO entries were identified. Each of these studies was manually examined to categorize the studies into bulk or single‐cell RNA‐seq (scRNA‐seq) followed by an inspection of their original articles. RESULTS: The currently available RNA‐seq data were generated from various types of patients’ samples, and COVID‐19 related sample materials have been sequenced at the level of RNA, including whole blood, different components of blood [e.g., plasma, peripheral blood mononuclear cells (PBMCs), leukocytes, lymphocytes, monocytes, T cells], nasal swabs, and autopsy samples (e.g., lung, heart, liver, kidney). Of these, RNA‐seq studies using whole blood, PBMCs, nasal swabs and autopsy/biopsy samples were reviewed to highlight the major findings from RNA‐seq data analysis. CONCLUSIONS: Based on the bulk and scRNA‐seq data analysis, severe COVID‐19 patients display shifts in cell populations, especially those of leukocytes and monocytes, possibly leading to cytokine storms and immune silence. These RNA‐seq data form the foundation for further gene expression analysis using samples from individuals suffering from long COVID