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RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq.

Polyphosphate (polyP) granule biogenesis is an ancient and ubiquitous starvation response in bacteria. Although the ability to make polyP is important for survival during quiescence and resistance to diverse environmental stresses, granule genesis is poorly understood. Using quantitative microscopy at high spatial and temporal resolution, we show that granule genesis in Pseudomonas aeruginosa is tightly organized under nitrogen starvation. Following nucleation as many microgranules throughout the nucleoid, polyP granules consolidate and become transiently spatially organized during cell cycle exit. Between 1 and 3 h after nitrogen starvation, a minority of cells have divided, yet the total granule number per cell decreases, total granule volume per cell dramatically increases, and individual granules grow to occupy diameters as large as ∼200 nm. At their peak, mature granules constitute ∼2% of the total cell volume and are evenly spaced along the long cell axis. Following cell cycle exit, granules initially retain a tight spatial organization, yet their size distribution and spacing relax deeper into starvation. Mutant cells lacking polyP elongate during starvation and contain more than one origin. PolyP promotes cell cycle exit by functioning at a step after DNA replication initiation. Together with the universal starvation alarmone (p)ppGpp, polyP has an additive effect on nucleoid dynamics and organization during starvation. Notably, cell cycle exit is temporally coupled to a net increase in polyP granule biomass, suggesting that net synthesis, rather than consumption of the polymer, is important for the mechanism by which polyP promotes completion of cell cycle exit during starvation.

Background Elevated serum free fatty acid (FFA) concentration is associated with insulin resistance and is a hallmark of metabolic syndrome. A pathological feature of insulin resistance is impaired endothelial function. Objective To investigate the effect of FFA reduction with either acipimox, a nicotinic acid derivative that impairs lipolysis, or salsalate, a salicylate that reduces basal and inflammation‐induced lipolysis, on insulin‐mediated endothelium‐dependent vasodilation. Methods This was a post hoc, combined analysis of two randomised, double‐blind, placebo‐controlled crossover trials. Sixteen subjects were recruited (6 with metabolic syndrome and 10 controls) and randomised to acipimox 250 mg orally every 6 h for 7 days or placebo. Nineteen subjects were recruited (13 with metabolic syndrome and 6 controls) and randomised to receive salsalate 4.5 g/day for 4 weeks or placebo. The primary outcome was the association between FFA concentration and insulin‐mediated vasodilation, measured by venous‐occlusion strain‐gauge plethysmography at baseline and following FFA modulation with the study drugs. Results At baseline, FFA concentration (R = −0.35, p = 0.043) and insulin sensitivity (HOMA‐IR: R = −0.42, p = 0.016, Adipo‐IR: R = −0.39, p = 0.025) predicted insulin‐mediated vasodilation. FFA levels were significantly reduced after drug pretreatment (0.604 vs. 0.491 mmol/L, p = 0.036) while insulin levels, insulin sensitivity and inflammatory markers were unchanged. Despite a reduction in circulating FFA with drug therapy, neither insulin‐stimulated vasodilation nor insulin sensitivity improved. Conclusions Short‐term reduction of FFA concentration does not improve insulin‐stimulated vasodilation in patients with metabolic syndrome. Trial Registration ClinicalTrials.gov identifier: NCT00759291 and NCT00760019 (formerly NCT00762827) At baseline, free fatty acid concentration and insulin sensitivity predicted insulin‐mediated vasodilation. Acipimox and salsalate reduced free fatty acid concentration, but did not change insulin sensitivity and there was no association between the change in free fatty acids and insulin‐mediated vasodilation.

Iron biofortified beans and carotenoid enriched cassava are proposed as a solution to combat iron and vitamin A deficiencies, respectively, in the Democratic Republic of Congo (DRC). To inform the need for biofortified foods, we conducted a survey in 2014 in two provinces of the DRC, South Kivu and Kongo Central. Unexpectedly, women of reproductive age (WRA; 15-49 y) and their children (6-59 m) had a low prevalence of biochemical iron and vitamin A deficiency, based on ferritin and retinol binding protein, respectively. To better understand the lack of biochemical deficiency of these nutrients, we examined the prevalence of inadequate intake for these and other select nutrients. Dietary intake was assessed using 24-hour recalls among 744 mother-child dyads. Repeat recalls on a non-consecutive day were conducted with a subsample of the study population to account for intra-individual variation and estimate usual intake. In WRA, the prevalence of inadequate iron intakes were 33% and 29% in South Kivu and Kongo Central, respecitvely. The prevalence of inadequate vitamin A intakes among WRA was low in South Kivu (18%) and negligible in Kongo Central (1%). Iron inadequacy was highest in infants (6-11 m) at 82% and 64% in South Kivu and Kongo Central, respectively. Among older children (12-59 m) in both provinces, the prevalence of iron inadequacy was similar at ~20%. There was a high prevalence of inadequate zinc intake in women and children (i.e. 79-86% among WRA and 56-91% among children 6-59 m) consistent with our findings of a high prevalence of low serum zinc in the same sample. Dietary data here corroborate the low prevalence of biochemical vitamin A deficiency but not iron. However, any change to the supply of red palm oil (primary source of vitamin A) would dramatically reduce population vitamin A intakes, thus a carotenoid enriched cassava program may be beneficial as a safety net measure. Crops biofortified with zinc also appear warranted. We caution that our findings cannot be extrapolated to the entire Congo where diverse agro-ecological landscape exist or when political and environmental shocks occur which challenge food production.

Iron biofortified beans and carotenoid enriched cassava are proposed as a solution to combat iron and vitamin A deficiencies, respectively, in the Democratic Republic of Congo (DRC). To inform the need for biofortified foods, we conducted a survey in 2014 in two provinces of the DRC, South Kivu and Kongo Central. Unexpectedly, women of reproductive age (WRA; 15-49 y) and their children (6-59 m) had a low prevalence of biochemical iron and vitamin A deficiency, based on ferritin and retinol binding protein, respectively. To better understand the lack of biochemical deficiency of these nutrients, we examined the prevalence of inadequate intake for these and other select nutrients. Dietary intake was assessed using 24-hour recalls among 744 mother-child dyads. Repeat recalls on a non-consecutive day were conducted with a subsample of the study population to account for intra-individual variation and estimate usual intake. In WRA, the prevalence of inadequate iron intakes were 33% and 29% in South Kivu and Kongo Central, respecitvely. The prevalence of inadequate vitamin A intakes among WRA was low in South Kivu (18%) and negligible in Kongo Central (1%). Iron inadequacy was highest in infants (6-11 m) at 82% and 64% in South Kivu and Kongo Central, respectively. Among older children (12-59 m) in both provinces, the prevalence of iron inadequacy was similar at ~20%. There was a high prevalence of inadequate zinc intake in women and children (i.e. 79-86% among WRA and 56-91% among children 6-59 m) consistent with our findings of a high prevalence of low serum zinc in the same sample. Dietary data here corroborate the low prevalence of biochemical vitamin A deficiency but not iron. However, any change to the supply of red palm oil (primary source of vitamin A) would dramatically reduce population vitamin A intakes, thus a carotenoid enriched cassava program may be beneficial as a safety net measure. Crops biofortified with zinc also appear warranted. We caution that our findings cannot be extrapolated to the entire Congo where diverse agro-ecological landscape exist or when political and environmental shocks occur which challenge food production.

Background Wetlands store a substantial amount of carbon (C) in deep soil organic matter deposits, and play an important role in global fluxes of carbon dioxide and methane. Fine roots (i.e., ephemeral roots that are active in water and nutrient uptake) are recognized as important components of biogeochemical cycles in nutrient-limited wetland ecosystems. However, quantification of fine-root dynamics in wetlands has generally been limited to destructive approaches, possibly because of methodological difficulties associated with the unique environmental, soil, and plant community characteristics of these systems. Non-destructive minirhizotron technology has rarely been used in wetland ecosystems. Scope Our goal was to develop a consensus on, and a methodological framework for, the appropriate installation and use of minirhizotron technology in wetland ecosystems. Here, we discuss a number of potential solutions for the challenges associated with the deployment of minirhizotron technology in wetlands, including minirhizotron installation and anchorage, capture and analysis of minirhizotron images, and upscaling of minirhizotron data for analysis of biogeochemical pools and parameterization of land surface models. Conclusions The appropriate use of minirhizotron technology to examine relatively understudied fineroot dynamics in wetlands will advance our knowledge of ecosystem C and nutrient cycling in these globally important ecosystems.