The ability to accurately quantify all the microRNAs (miRNAs) in a sample is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. We develop a new method for preparing miRNA sequencing libraries, RealSeq®-AC, that involves ligating the miRNAs with a single adapter and circularizing the ligation products. When compared to other methods, RealSeq®-AC provides greatly reduced miRNA sequencing bias and allows the identification of the largest variety of miRNAs in biological samples. This reduced bias also allows robust quantification of miRNAs present in samples across a wide range of RNA input levels.Electronic supplementary materialThe online version of this article (10.1186/s13059-018-1488-z) contains supplementary material, which is available to authorized users.

Benign prostatic hyperplasia, prostate cancer, and changes in the ratio of circulating testosterone and estradiol often occur concurrently in aging men and can lead to lower urinary tract (LUT) dysfunction. To explore the possibility of a fetal basis for the development of LUT dysfunction in adulthood, Tg(CMV-cre);Nkx3-1(+/-);Pten(fl/+) mice, which are genetically predisposed to prostate neoplasia, were exposedin uteroand during lactation to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 1 μg/kg po) or corn oil vehicle (5 ml/kg) after a single maternal dose on 13 days post coitus, and subsequently were aged without further manipulation, or at 8 weeks of age were exposed to exogenous 17 β-estradiol (2.5 mg) and testosterone (25 mg) (T+E2) via slow release subcutaneous implants.In uteroand lactational (IUL) TCDD exposure in the absence of exogenous hormone treatment reduced voiding pressure in adult mice, but otherwise had little effect on mouse LUT anatomy or function. By comparison, IUL TCDD exposure followed by exogenous hormone treatment increased relative kidney, bladder, dorsolateral prostate, and seminal vesicle weights, hydronephrosis incidence, and prostate epithelial cell proliferation, thickened prostate periductal smooth muscle, and altered prostate and bladder collagen fiber distribution. We propose a 2-hit model whereby IUL TCDD exposure sensitizes mice to exogenous-hormone-induced urinary tract dysfunction later in life.

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3′ nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3′ ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3′ ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3′ end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores.

It is tragic and ironic, as we speak of the pandemic of physical inactivity, that we already know the cure for physical inactivity, the 4 th leading risk factor for mortality costing billions of dollars in medically related costs and losses in productivity. The solution is simple. People must move more often. And of exceptional relevance, physical activity can prevent diseases which increase the population's susceptibility to the new coronavirus pandemic, CO-VID-19. Creating innovative programs which encourage movement is a beginning, but these programs must be sustainable and accessible to a country's vulnerable populations. 3 WINS Fitness is a free scalable and innovative community-based exercise program serving over 300 participants requiring no external funding for daily operations due to its implementation by university kinesiology students. If we apply our knowledge and work together in significant collaborations, millions of lives can be saved. Population physical activity has not increased since the late 1990's. We must take a fresh look at identifying new or unique collaboratives and re-inventing current systems. At the core is the education system of university kinesiology/exercise science programs, teaching students the complete landscape of what is required for increases in population physical activity. The Call to Action (CTA) is kinesiology/exercise science departments around the world vigorously taking on the challenge and owning the responsibility for increasing population physical activity. The students of today can control the health destiny of millions of people around the world. The first steps to these departments taking the lead must begin today.

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3′ nucleotidyl transferases, S. cerevisiae poly(A) polymerase and C. elegans Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3′ ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3′ ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3′ end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores.