How the distribution of anthropogenic nitrogen has changed in Narragansett Bay (RI, USA) following major reductions in nutrient loads.

Over the past decade, nitrogen (N) loads to Narragansett Bay have decreased by more than 50%. These reductions were, in large part, the direct result of multiple wastewater treatment facility upgrades to tertiary treatment, a process which employs N removal. Here we document ecosystem response to the N reductions and assess how the distribution of sewage N in Narragansett Bay has changed from before, during, and shortly after the upgrades. While others have observed clear responses when data were considered annually, our seasonal and regional comparisons of pre- and post-tertiary treatment dissolved inorganic nitrogen (DIN) concentrations and Secchi depth data, from bay-wide surveys conducted periodically from the early 1970s through 2016, resulted in only a few subtle differences. Thus we sought to use stable isotope data to assess how sewage N is incorporated into the ecology of the Bay and how its distribution may have changed after the upgrades. The nitrogen (δ15N) and carbon (δ13C) stable isotope measurements of particulate matter served as a proxy for phytoplankton, while macroalgae served as short-term integrators of water column bio-available N, and hard clams (Mercenaria mercenaria) as integrators of water column production. In contrast to other estuarine stable isotope studies that have observed an increased influence of isotopically lower marine N when sewage N is reduced, the opposite has occurred in Narragansett Bay. The tertiary treatment upgrades have increased the effluent δ15N values by at least 2‰. The plants and animals throughout Narragansett Bay have similarly increased by 1-2‰, on average. In contrast, the δ13C values measured in particulate matter and hard clams have declined by about the same amount. The δ15N results indicated that, even after the N-reductions, sewage N still plays an important role in supporting primary and secondary production throughout the Bay. However, the δ13C suggest that overall net production in Narragansett Bay has decreased. In the five years after the major wastewater treatment facilities came on-line for nutrient removal, oligotrophication has begun but sewage remains the dominant source of N to Narragansett Bay.

Changes to nitrate isotopic composition of wastewater treatment effluent and rivers after upgrades to tertiary treatment in the Narragansett Bay watershed, RI.

Due to nitrogen load reduction policies, wastewater treatment facilities (WWTFs) have upgraded to tertiary treatment - where denitrification reduces and removes nitrogen. Changes to the stable isotopic composition of nitrate inputs after upgrades or how it transfers to the estuary have not been assessed in Rhode Island. We investigate whether these upgrades impact the isotopic signature of nitrate inputs to Narragansett Bay. Samples from rivers and WWTFs discharging to Narragansett Bay characterize the anthropogenic source nitrate (NO3(-)) isotopic composition (δ(15)N-NO3(-) and δ(18)O-NO3(-)) and temporal variability. At one WWTF, tertiary treatment increased effluent nitrate δ(15)N-NO3(-) and δ(18)O-NO3(-) values by ~16‰. Riverine values increased by ~4‰, likely due to the combination of decreases in N and upgrades. Combined river and WWTF flux-weighted isotopic compositions showed enriched values and an amplitude reduction in monthly variability. When seasonal isotopic means are significantly different from other sources, δ(15)N-NO3(-) may be a useful tracer of inputs.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.