N6-methyladenosine (m6A) is one of the most abundant messenger RNA modifications in eukaryotes involved in various pivotal processes of RNA metabolism. The most popular high-throughput m6A identification method depends on the anti-m6A antibody but suffers from poor reproducibility and limited resolution. Exact location information is of great value for understanding the dynamics, machinery, and functions of m6A. Here, we developed a precise and high-throughput antibody-independent m6A identification method based on the m6A-sensitive RNA endoribonuclease recognizing ACA motif (m6A-sensitive RNA-Endoribonuclease–Facilitated sequencing or m6A-REF-seq). Whole-transcriptomic, single-base m6A maps generated by m6A-REF-seq quantitatively displayed an explicit distribution pattern with enrichment near stop codons. We used independent methods to validate methylation status and abundance of individual m6A sites, confirming the high reliability and accuracy of m6A-REF-seq. We applied this method on five tissues from human, mouse, and rat, showing that m6A sites are conserved with single-nucleotide specificity and tend to cluster among species.

A strict transfusion algorithm can reduce the transfusion requirement for all blood components. Preheparin testing of platelet function with adenosine diphosphate aggregometry can identify patients at highest risk for perioperative bleeding and transfusions and might further reduce the perioperative transfusion requirement.

Evidence suggests that the sympathetic nervous system and ␣ 1 -adrenoreceptors (AR) 1 may exert trophic influences over SMCs during normal development and also contribute to the pathogenesis of vascular hypertrophy and atherosclerosis (1, 2). Hyperinnervation of blood vessels by catecholaminergic fibers in the genetic spontaneously hypertensive rat has been correlated with SMC hypertrophy and hyperplasia in these vessels (3). Also, sympathectomy attenuates normal growth, as well as hypertrophy of the vascular wall in hypertensive animals (4 -7). There is considerable evidence that smoking, stress, and hypertension, which are key risk factors for atherosclerosis and hypertrophic vascular disease, are associated with elevated plasma catecholamines (8, 9).Catecholamines have been shown to initiate not only immediate SMC responses such as contraction of blood vessels, but may also influence proliferation and growth of cultured vascular SMCs (10 -13). In nonconfluent, cultured rat and rabbit aortic SMCs, AR stimulation promotes cell proliferation (10, 13). Furthermore, ␣ 1 blockade reduces vascular collagen synthesis in the spontaneous hypertensive rat (14), and inhibits SMC proliferation induced by endothelial denudation (13, 15) and angiotensin infusion (16) in normal rats. In cholesterol-fed monkeys, elevated plasma norepinephrine (NE) greatly increased atherosclerotic lesion growth (17). Infusion of NE over a 2-week period, at a level which did not cause sustained elevation of blood pressure, induced formation of atherosclerotic vascular lesions in rabbit aorta (18). There is also evidence that ␣ 1 ARs mediate growth of myocardial cells. In cultured neonatal rat cardiac myocytes that possess both ␤ 1 and ␣ 1 ARs, stimulation of ␣ 1 ARs with NE increased cell protein, RNA, myocyte surface area, and contractile protein expression (19). However, no studies have examined whether ␣ 1 ARs influence proliferation-independent growth of SMCs.Both molecular cloning and pharmacologic studies have shown that ␣ 1 ARs are comprised of three closely related subtypes (20, 21). We have recently used polymerase chain reaction (22) and RNase protection assays 2 to determine expression of ␣AR subtype mRNA by rat vascular SMCs. Freshly isolated and early passage cultured aortic and vena cava SMCs express both ␣ 1B and ␣ 1D mRNA (20), and it appears that both receptors are present on SMCs of rat aorta (24,25, and Ref. 22 and references therein). Rat aorta has been shown recently to also express ␣ 1A (formerly denoted "␣ 1C ") mRNA (26 -29). Given this multiplicity of ␣ 1 AR expression, the purpose of the present study was first to examine both in vitro and in situ, the effects of combined stimulation of the ␣ 1 AR subtypes with NE alone on proliferation-independent growth, and expression of sarcomeric ␣-SMC-actin and cytoskeletal ␤-actin mRNAs by arterial and venous SMCs. Second, effects of combined stimulation were compared with those during treatment with NE plus antagonists, 5-methylurapidil (5-MU, selectivity ϭ ␣ 1A Ͼ ␣ 1D Ͼ ␣ 1B...

No abstract

No abstract