Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More.

The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called "fluorogens" that become fluorescent upon binding to genetically encodable RNAs called "light-up aptamers." Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.journal article2020importe

Correction to: Cluster identification, selection, and description in Cluster randomized crossover trials: the PREP-IT trials

An amendment to this paper has been published and can be accessed via the original article

Low Na, high K diet and the role of aldosterone in BK-mediated K excretion.

A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-β4; BK-α/β4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/β4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-β4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-β4 knockout (β4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of β4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in β4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/β4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease

The effects of ADX and aldo replacement on K balance of LNaHK mice.

<p><b>(A)</b> The rate of urinary K excretion (U_KV; * <i>P</i> < 0.001 vs. sham; # <i>P</i> < 0.001 vs. ADX-LA), <b>(B)</b> Transtubular K gradient (TTKG; * <i>P</i> = 0.002 vs. sham; ^#<i>P</i> = 0.002 vs. ADX-LA), and <b>(C)</b> Urinary flow (* <i>P</i> < 0.001 vs. sham; # <i>P</i> < 0.001 vs. ADX-LA) in sham (<i>n</i> = 7), ADX-LA (<i>n</i> = 6), and ADX-HA (<i>n</i> = 7) mice.</p

Characteristics of β4KO LNaHK sham, ADX-LA, and ADX-HA mice.

<p>Abbreviations: LNaHK, low Na, high K; ADX-LA, adrenalectomy with low dose aldosterone replacement; ADX-HA, adrenalectomy with high dose aldosterone; SEM, standard error of mean; Hct, hematocrit; BUN, blood urea nitrogen; P[aldo]; plasma [aldosterone]. Sham, <i>n</i> = 7; ADX-LA, <i>n</i> = 5; ADX-HA, <i>n</i> = 5.</p><p>Characteristics of β4KO LNaHK sham, ADX-LA, and ADX-HA mice.</p

The effect of high K diets, adrenalectomy (ADX), and aldo replacement on plasma aldosterone (aldo) concentration (P[aldo]).

<p><b>(A)</b> Bar plot of P[aldo] in sham mice on control (Con; <i>n</i> = 5), high K (HK; <i>n</i> = 7), and low Na, high K (LNaHK; <i>n</i> = 6) diets. * <i>P</i> < 0.001 vs. Con; # <i>P</i> < 0.001 vs. HK. <b>(B)</b> Bar plot of P[aldo] in sham (<i>n</i> = 4) and ADX mice receiving a low dose replacement of aldo (ADX-LA; <i>n</i> = 6), and ADX mice receiving high dose aldo (ADX-HA; <i>n</i> = 7). * <i>P</i> < 0.001 vs. sham; # <i>P</i> < 0.001 vs. ADX-LA.</p

The effect of aldo on β4KO LNaHK mice.

<p>The K clearance <b>(A)</b> and TTKG <b>(B)</b> of WT and β4KO mice on Con given sham or ADX surgery or on LNaHK given sham, ADX-LA, or ADX-HA. * <i>P</i> < 0.05 vs. WT. <b>(C)</b> The relation between P[aldo] and U_KV determined in WT and β4KO ADX mice receiving various doses of aldo replacement.</p

The effects of amiloride on osmolar clearance.

<p>The osmolar clearance was determined in WT Con (<i>n</i> ≥ 5), WT LNaHK (<i>n</i> = 9), and β4KO LNaHK (<i>n</i> ≥ 5) mice given vehicle or amiloride. * <i>P</i> < 0.05 vs. vehicle; # <i>P</i> < 0.05 vs. WT LNaHK.</p

Characteristics of WT LNaHK sham, ADX-LA, and ADX-HA mice.

<p>Abbreviations: LNaHK, low Na, high K; ADX-LA, adrenalectomy with low dose aldosterone replacement; ADX-HA, adrenalectomy with high dose aldosterone; SEM, standard error of mean; Hct, hematocrit; BUN, blood urea nitrogen; P[aldo]; plasma [aldosterone]. Sham, <i>n</i> = 7; ADX-LA, <i>n</i> = 6; ADX-HA, <i>n</i> = 7.</p><p>Characteristics of WT LNaHK sham, ADX-LA, and ADX-HA mice.</p

The effect of aldo on BK-α expression in the distal nephron of LNaHK mice.

<p>Representative images <b>(A)</b> and bar plots summarizing intensity of fluorescent immunohistochemical staining of apical <b>(B)</b> and total <b>(C)</b> BK-α in cortical kidney sections of sham, ADX-LA, and ADX-HA mice. BK-α (green) was co-stained with V-ATPase (red), a marker of intercalated cells in the connecting tubule and collecting duct. Merged images also contain Hoechst nuclear stain (blue). * <i>P</i> < 0.001 vs. sham; # <i>P</i> < 0.001 vs. ADX-LA; <i>n</i> = 9.</p