Direct identification of A-to-I editing sites with nanopore native RNA sequencing.

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.

Deep transcriptome profiling reveals limited conservation of A-to-I RNA editing in Xenopus.

BACKGROUND: Xenopus has served as a valuable model system for biomedical research over the past decades. Notably, ADAR was first detected in frog oocytes and embryos as an activity that unwinds RNA duplexes. However, the scope of A-to-I RNA editing by the ADAR enzymes in Xenopus remains underexplored. RESULTS: Here, we identify millions of editing events in Xenopus with high accuracy and systematically map the editome across developmental stages, adult organs, and species. We report diverse spatiotemporal patterns of editing with deamination activity highest in early embryogenesis before zygotic genome activation and in the ovary. Strikingly, editing events are poorly conserved across different Xenopus species. Even sites that are detected in both X. laevis and X. tropicalis show largely divergent editing levels or developmental profiles. In protein-coding regions, only a small subset of sites that are found mostly in the brain are well conserved between frogs and mammals. CONCLUSIONS: Collectively, our work provides fresh insights into ADAR activity in vertebrates and suggest that species-specific editing may play a role in each animal's unique physiology or environmental adaptation.

Matrix mechanotransduction mediated by thrombospondin-1/integrin/YAP in the vascular remodeling.

The extracellular matrix (ECM) initiates mechanical cues that activate intracellular signaling through matrix-cell interactions. In blood vessels, additional mechanical cues derived from the pulsatile blood flow and pressure play a pivotal role in homeostasis and disease development. Currently, the nature of the cues from the ECM and their interaction with the mechanical microenvironment in large blood vessels to maintain the integrity of the vessel wall are not fully understood. Here, we identified the matricellular protein thrombospondin-1 (Thbs1) as an extracellular mediator of matrix mechanotransduction that acts via integrin αvß1 to establish focal adhesions and promotes nuclear shuttling of Yes-associated protein (YAP) in response to high strain of cyclic stretch. Thbs1-mediated YAP activation depends on the small GTPase Rap2 and Hippo pathway and is not influenced by alteration of actin fibers. Deletion of Thbs1 in mice inhibited Thbs1/integrin ß1/YAP signaling, leading to maladaptive remodeling of the aorta in response to pressure overload and inhibition of neointima formation upon carotid artery ligation, exerting context-dependent effects on the vessel wall. We thus propose a mechanism of matrix mechanotransduction centered on Thbs1, connecting mechanical stimuli to YAP signaling during vascular remodeling in vivo.

The Use of a Production Function to Evaluate Caribbean Ocean Health Conflicts and Economic Development: Policy Implications.

Central American and Caribbean (CAC) countries enjoy diverse marine environments. The oceans that enclose these coastlines contribute significantly to their economic growth. Industrial expansion and tourism place pressure on the marine ecosystems causing a decline in ocean health. To evaluate the cause of ocean health changes we investigated the factors influencing CAC ocean health using a health production function. Using Rank-Based Regression and the set of extractive, cultural and human well-being and services goals measuring Ocean Health Index (OHI), data from the World Bank, and the Human Development Index we developed a production function for CAC countries ocean health. Results show that all regional OHI scores, but Tourism and Recreation, the main income earning industry for most of the CAC countries, are less than the global score with four of the goals less than 40. The production function shows that all the goals, but the biodiversity sub-goal species, positively influencing OHI. Two climatic related variables, Nitrous Oxide and Carbon Dioxide negatively influenced OHI. The results are important to policy makers as they decide on the need to make greater effort towards improving sustainable contribution of CAC ocean resources to the blue economy.

Protective Role of Endothelial Fibulin-4 in Valvulo-Arterial Integrity.

Background Homeostasis of the vessel wall is cooperatively maintained by endothelial cells (ECs), smooth muscle cells, and adventitial fibroblasts. The genetic deletion of fibulin-4 (Fbln4) in smooth muscle cells (SMKO) leads to the formation of thoracic aortic aneurysms with the disruption of elastic fibers. Although Fbln4 is expressed in the entire vessel wall, its function in ECs and relevance to the maintenance of valvulo-arterial integrity are not fully understood. Methods and Results Gene silencing of FBLN4 was conducted on human aortic ECs to evaluate morphological changes and gene expression profile. Fbln4 double knockout (DKO) mice in ECs and smooth muscle cells were generated and subjected to histological analysis, echocardiography, Western blotting, RNA sequencing, and immunostaining. An evaluation of the thoracic aortic aneurysm phenotype and screening of altered signaling pathways were performed. Knockdown of FBLN4 in human aortic ECs induced mesenchymal cell-like changes with the upregulation of mesenchymal genes, including TAGLN and MYL9. DKO mice showed the exacerbation of thoracic aortic aneurysms when compared with those of SMKO and upregulated Thbs1, a mechanical stress-responsive molecule, throughout the aorta. DKO mice also showed progressive aortic valve thickening with collagen deposition from postnatal day 14, as well as turbulent flow in the ascending aorta. Furthermore, RNA sequencing and immunostaining of the aortic valve revealed the upregulation of genes involved in endothelial-to-mesenchymal transition, inflammatory response, and tissue fibrosis in DKO valves and the presence of activated valve interstitial cells. Conclusions The current study uncovers the pivotal role of endothelial fibulin-4 in the maintenance of valvulo-arterial integrity, which influences thoracic aortic aneurysm progression.

Fluorescence microscopy datasets for training deep neural networks.

BACKGROUND: Fluorescence microscopy is an important technique in many areas of biological research. Two factors that limit the usefulness and performance of fluorescence microscopy are photobleaching of fluorescent probes during imaging and, when imaging live cells, phototoxicity caused by light exposure. Recently developed methods in machine learning are able to greatly improve the signal-to-noise ratio of acquired images. This allows researchers to record images with much shorter exposure times, which in turn minimizes photobleaching and phototoxicity by reducing the dose of light reaching the sample. FINDINGS: To use deep learning methods, a large amount of data is needed to train the underlying convolutional neural network. One way to do this involves use of pairs of fluorescence microscopy images acquired with long and short exposure times. We provide high-quality datasets that can be used to train and evaluate deep learning methods under development. CONCLUSION: The availability of high-quality data is vital for training convolutional neural networks that are used in current machine learning approaches.

Contribution of PDGFRα-positive cells in maintenance and injury responses in mouse large vessels.

The maladaptive remodeling of vessel walls with neointima formation is a common feature of proliferative vascular diseases. It has been proposed that neointima formation is caused by the dedifferentiation of mature smooth muscle cells (SMCs). Recent evidence suggests that adventitial cells also participate in neointima formation; however, their cellular dynamics are not fully understood. In this study, we utilized a lineage tracing model of platelet-derived growth factor receptor alpha (PDGFRa) cells and examined cellular behavior during homeostasis and injury response. PDGFRa marked adventitial cells that were largely positive for Sca1 and a portion of medial SMCs, and both cell types were maintained for 2 years. Upon carotid artery ligation, PDGFRa-positive (+) cells were slowly recruited to the neointima and exhibited an immature SMC phenotype. In contrast, in a more severe wire denudation injury, PDGFRa+ cells were recruited to the neointima within 14 days and fully differentiated into SMCs. Under pressure overload induced by transverse aortic constriction, PDGFRa+ cells developed marked adventitial fibrosis. Taken together, our observations suggest that PDGFRa+ cells serve as a reservoir of adventitial cells and a subset of medial SMCs and underscore their context-dependent response to vascular injuries.