The use of antibiotic resistance analysis (ARA) for microbial source tracking requires the generation of a library of isolates collected from known sources in the watershed. The size and composition of the library are critical in determining if it represents the diversity of patterns found in the watershed. This study was performed to determine the size that an ARA library needs to be to be representative of the watersheds for which it will be used and to determine if libraries from different watersheds can be merged to create multiwatershed libraries. Fecal samples from known human, domesticated, and wild animal sources were collected from six Virginia watersheds. From these samples, enterococci were isolated and tested by ARA. Based on cross-validation discriminant analysis, only the largest of the libraries (2,931 isolates) were found to be able to classify nonlibrary isolates as well as library isolates (i.e., were representative). Small libraries tended to have higher average rates of correct classification, but were much less able to correctly classify nonlibrary isolates. A merged multiwatershed library (6,587 isolates) was created and was found to be large enough to be representative of the isolates from the contributing watersheds. When isolates that were collected from the contributing watersheds approximately 1 year later were analyzed with the multiwatershed library, they were classified as well as the isolates in the library, suggesting that the resistance patterns are temporally stable for at least 1 year. The ability to obtain a representative, temporally stable library demonstrates that ARA can be used to identify sources of fecal pollution in natural waters.

[1] The global Boron (B) cycle is primarily driven by a large flux (1.44 Tg B/yr) through the atmosphere derived from seasalt aerosols. Other significant sources of atmospheric boron include emissions during the combustion of biomass (0.26-0.43 Tg B/yr) and coal, which adds 0.20 Tg B/yr as an anthropogenic contribution. These known inputs to the atmosphere cannot account for the boron removed from the atmosphere during rainfall (3.0 Tg B/yr) and estimated dry deposition (1.3-2.7 Tg B/yr). In addition to atmospheric deposition, rock weathering is a source of boron (0.19 Tg B/yr) for terrestrial ecosystems, and humans mine about 0.31 Tg B/yr from the Earth's crust. More than 4.8 Tg B/yr circulates in the biogeochemical cycle of land plants, and about 0.53-0.63 Tg B/yr is carried from land to sea by rivers. The biogeochemical cycle of boron in the sea includes 4.4 Tg B/yr circulating in the marine biosphere, and an annual loss of 0.47 Tg B/yr to the oceanic crust via a variety of sedimentary processes that collectively remove only a small fraction of the total annual inputs to the oceans. Thus with our current understanding of the global biogeochemistry of B, the atmospheric budget shows outputs > inputs, while the marine compartments show inputs > outputs. Despite these uncertainties, it is clear that the human perturbation of the global B cycle has more than doubled the mobilization of B from the crust and contributes significantly to the B transport in rivers.

Autophagy is a homeostatic process that is important for degrading protein aggregates, nutrient deposits, dysfunctional organelles and several signaling molecules. p62/sequestosome-1 is a protein that binds to several autophagy substrates, such as ubiquitinated proteins, damaged mitochondria and signaling molecules such as an Nrf2 inhibitor Keap1, promoting their autophagic degradation. Sestrin2, a stress-inducible protein, has been recently shown to bind to p62 and promote autophagic degradation of such p62 targets. Because Sestrin2 is a metabolic regulator that suppresses diverse age- and obesity-associated pathologies, the autophagy-controlling function of Sestrin2 may be important for its other physiological functions. However, the molecular mechanism of how Sestrin2 can promote clearance of p62-associated proteins has been unclear. Here we show that Sestrin2 physically associates with ULK1 and p62 to form a complex, in which both Sestrin2 and p62 become phosphorylated by ULK1 at multiple sites. Ser403 of p62, whose phosphorylation is known to promote autophagic degradation of p62 and its targets, is among the sites phosphorylated by ULK1. ULK1-mediated p62 phosphorylation was facilitated by Sestrin2 in cells as well as in vitro kinase assays. Consistent with this finding, oligomycin-induced energy deprivation, which strongly activates ULK1, provoked a robust Ser403 phosphorylation of p62 in wild-type (WT) mouse embryonic fibroblasts (MEF). However, in ULK1/2- and Sestrin2-deficient MEF, oligomycin-induced p62 phosphorylation was dramatically attenuated, suggesting that endogenous Sestrin2-ULK1/2 mainly mediates p62 phosphorylation in response to energetic stress. Taken together, this study identifies ULK1 as a new p62 Ser403 kinase and establishes Sestrin2 as a promoter of ULK1-mediated p62 phosphorylation.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2 −/− mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold-or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROSmediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.aging | mouse | homeostasis | β-adrenergic signaling A lthough reactive oxygen species (ROS) are normal products of cellular metabolism, excessive accumulation of ROS resulting from nutritional imbalance and/or environmental stresses can provoke oxidative damage of diverse cellular macromolecules, such as DNA, RNA, and proteins (1). Accumulation of ROS has been associated with diverse degenerative diseases, such as cancer, neurodegeneration, and obesity-associated metabolic syndrome (2-4). To minimize detrimental consequences of ROS accumulation, cells are equipped with various antioxidant proteins, including superoxide dismutases, catalases, peroxiredoxins, and sestrins (5-7). Several ROS-scavenging chemicals or dietary supplements, such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), and antioxidant vitamins, can assist with eliminating excessive amounts of ROS (8-10) and were once considered to be potential inhibitors of degenerative diseases associated with aging and obesity (11-13). However, most animal and human clinical studies failed to demonstrate the benefits of dietary antioxidants in restoring metabolic homeostasis or in promoting health and lifespan (13,14).Uncoupling protein 1 (Ucp1) is an anion-carrier protein located in the inner membrane of the mitochondria. By dissipatin...