Bioluminescence resonance energy transfer (BRET) from an exceptionally bright luciferase, NanoLuc, to a fluorescent HaloTag ligand is gaining momentum to monitor molecular interactions. The recommended use of HaloTag618 ligand for the NanoLuc-HaloTag BRET pair is versatile for ensemble experiments due to their well-separated emission bands. However, this system is not applicable for single-cell BRET imaging because of its low BRET efficiency and in turn weak acceptor signals.Here we explored the unprecedented potential of rhodamine based HaloTag ligands, containing azetidine rings, as BRET acceptors. Through a comprehensive evaluation of various commercial and Janelia Fluor HaloTag ligands for improved BRET efficiency and minimal donor signal bleed-through, we identified JF525 to be the best acceptor for microscopic BRET imaging. We successfully employed BRET imaging with JF525 to monitor the interaction of protein kinase A catalytic and regulatory subunit. Single-cell BRET imaging with HaloTag JF525 can henceforth open doors to comprehend and interpret molecular interactions.
Background The IGF2 (insulin-like growth factor 2) and H19 gene cluster plays an important role during pregnancy as it promotes both foetal and placental growth. We investigated the association between cord blood DNA methylation status of the IGF2/H19 gene cluster and maternal fine particulate matter exposure during fetal life. To the best of our knowledge, this is the first study investigating the association between prenatal PM2.5 exposure and newborn DNA methylation of the IGF2/H19. Methods Cord blood DNA methylation status of IGF2/H19 cluster was measured in 189 mother-newborn pairs from the ENVIRONAGE birth cohort (Flanders, Belgium). We assessed the sex-specific association between residential PM2.5 exposure during pregnancy and the methylation level of CpG loci mapping to the IGF2/H19 cluster, and identified prenatal vulnerability by investigating susceptible time windows of exposure. We also addressed the biological functionality of DNA methylation level in the gene cluster. Results Prenatal PM2.5 exposure was found to have genetic region-specific significant association with IGF2 and H19 during specific gestational weeks. The association was found to be sex-specific in both gene regions. Functionality of the DNA methylation was annotated by the association to fetal growth and cellular pathways. Conclusions The results of our study provided evidence that prenatal PM2.5 exposure is associated with DNA methylation in newborns’ IGF2/H19. The consequences within the context of fetal development of future phenotyping should be addressed.
There is an increasing interest in microRNAs (miRNAs) as they are of utmost importance in gene regulation at the posttranscriptional level. Sex-related susceptibility for non-communicable diseases later in life could originate in early life. Until now, no data on sex-specific miRNA expression are available for the placenta. Therefore, we investigated the difference by sex of newborn's miRNA expression in human placental tissue. Within the ENVIRONAGE birth cohort, miRNA and mRNA expression profiling was performed in 60 placentae (50% boys) using Agilent (8 × 60 K) microarrays. The distribution of chromosome locations was studied and pathway analysis of the identified sex-specific miRNAs in the placenta was carried out. Of the total 2558 miRNAs on the array, 597 miRNAs were expressed in over 70% of the samples and were included for further analyses. A total of 142 miRNAs were significantly (FDR<0.05) associated with the newborn's sex. In newborn girls, 76 miRNAs had higher expression (hsa-miR-361-5p as most significant) and 66 miRNAs had lower expression (hsa-miR-4646-5p as most significant) than in newborn boys. In the same study population, placental differentially expressed genes by sex were also identified using a whole genome approach. The placental gene expression revealed 27 differentially expressed genes by comparing girls to boys. Ultimately, we studied the miRNA-RNA interactome and identified 14 miRNA-mRNA interactions as sex-specific. Sex differences in placental m(i)RNA expression may reveal sex-specific patterns already present during pregnancy, which may influence physiological conditions in early or later life. These molecular processes might play a role in sex-specific disease susceptibility in later life.
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