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BACKGROUND: Annually, approximately 15 million babies are born preterm (< 37 weeks gestational age) globally. In the neonatal intensive care unit (NICU) environment, infants are exposed to repeated stressful or painful procedures as part of routine lifesaving care. These procedures have been associated with epigenetic alterations that may lead to an increased risk of neurodevelopmental disorders. Telomere length has been negatively associated with adverse life experiences in studies of adults. OBJECTIVES: This pilot study aimed to describe telomere length in a sample of preterm infants at NICU discharge and examine any associations with pain, feeding method, and neurodevelopment. METHODS: This descriptive pilot study sample includes baseline absolute telomere length (aTL) of 36 preterm infants immediately prior to discharge. Quantitative polymerase chain reaction (qPCR) was used to determine absolute telomere length. Infant demographics, pain/stress, type of feeding, antibiotic use, neurodevelopment, and buccal swab data were collected. Descriptive data analysis was used to describe the telomere length using graphs. RESULTS: Among our preterm infant samples, the mean absolute telomere length was far greater than the average adult telomere length. While no significant associations were found between absolute telomere length and pain, feeding method, and neurodevelopment, a trend between sex was noted where male telomere lengths were shorter than females as they aged. DISCUSSION: This is one of few studies to evaluate preterm infant telomere length. While other researchers have used relative telomere length, we used the more accurate absolute telomere length. We found nonsignificant shorter telomere lengths among males. Additional large-scale, longitudinal studies are needed to better identify the predictors of telomere length at the time of discharge from NICU.

The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and –EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.

Endothelial cells are important contributors to brain development, physiology, and disease. Although RNA sequencing has contributed to the understanding of brain endothelial cell diversity, bulk analysis and single-cell approaches have relied on fresh tissue digestion protocols for the isolation of single endothelial cells and flow cytometry-based sorting on surface markers or transgene expression. These approaches are limited in the analysis of the endothelium in human brain tissues, where fresh samples are difficult to obtain. Here, we developed an approach to examine endothelial RNA expression by using an endothelial-specific marker to isolate nuclei from abundant archived frozen brain tissues. We show that this approach rapidly and reliably extracts endothelial nuclei from frozen mouse brain samples, and importantly, from archived frozen human brain tissues. Furthermore, isolated RNA transcript levels are closely correlated with expression in whole cells from tissue digestion protocols and are enriched in endothelial markers and depleted of markers of other brain cell types. As high-quality RNA transcripts could be obtained from as few as 100 nuclei in archived frozen human brain tissues, we predict that this approach should be useful for both bulk analysis of endothelial RNA transcripts in human brain tissues as well as single-cell analysis of endothelial sub-populations.