Beyond Old Pipes and Ailing Budgets: Systems Thinking on Twenty-First Century Water Infrastructure in Chicago.

Cities are increasingly burdened by aging water infrastructure. Deferred maintenance and upgrades are compounded by emerging concerns over contaminants, extreme weather events, demographic shifts, equity, and affordability of water services. These and other evolving twenty-first century conditions prompt changes to urban water infrastructure and related systems that have wide ranging outcomes. This work demonstrates two complementary techniques for analyzing these complex systems, through the example case of Chicago. Chicago has some of the oldest urban water infrastructure in the US and supplies drinking water to more than 5 million people. Recent efforts to improve the physical and financial components of Chicago's water system have run into a gamut of social and environmental issues. Here, a socio-environmental systems (SES) context for Chicago's water infrastructure is structured using a rigorous systems thinking method and visual grammar to map the SES in terms of distinctions, systems, relationships and perspectives (DSRP). DSRP maps structure information about how water flows through city and how money flows through the public utilities responsible for drinking water delivery, wastewater treatment and stormwater management. Flows are evaluated, using open data and methods, over a 23-year period (1995-2017). Overall declines in water use and wastewater production are accompanied by an increase in the costs of water services, costs that support not only water infrastructure operations, maintenance and capital improvements, but also other municipal functions. Trends in the integrated data are interpreted through iterative refinement of DSRP maps to include additional components and to consider the SES from different points of view. Findings suggest that systems thinking is important for designing urban water system upgrades that are responsive to diverse socio-environmental concerns. As changes are made, transparent, reproducible methods for tracking outcomes can support analysis of differential impacts on users. The methods applied here at the city scale may be used to better understand localized, complex issues surrounding water infrastructure upgrades in Chicago and other cities.

Microfluidic chambers using fluid walls for cell biology.

Many proofs of concept have demonstrated the potential of microfluidics in cell biology. However, the technology remains inaccessible to many biologists, as it often requires complex manufacturing facilities (such as soft lithography) and uses materials foreign to cell biology (such as polydimethylsiloxane). Here, we present a method for creating microfluidic environments by simply reshaping fluids on a substrate. For applications in cell biology, we use cell media on a virgin Petri dish overlaid with an immiscible fluorocarbon. A hydrophobic/fluorophilic stylus then reshapes the media into any pattern by creating liquid walls of fluorocarbon. Microfluidic arrangements suitable for cell culture are made in minutes using materials familiar to biologists. The versatility of the method is demonstrated by creating analogs of a common platform in cell biology, the microtiter plate. Using this vehicle, we demonstrate many manipulations required for cell culture and downstream analysis, including feeding, replating, cloning, cryopreservation, lysis plus RT-PCR, transfection plus genome editing, and fixation plus immunolabeling (when fluid walls are reconfigured during use). We also show that mammalian cells grow and respond to stimuli normally, and worm eggs develop into adults. This simple approach provides biologists with an entrée into microfluidics.

An R Package for Open, Reproducible Analysis of Urban Water Systems, With Application to Chicago.

Urban water systems consist of natural and engineered flows of water interacting in complex ways. System complexity can be understood via mass conservative models that account for the interrelationships among all major flows and storages. We have developed a generic urban water system model in the R package CityWaterBalance. CityWaterBalance provides a reproducible workflow for studying urban water systems by facilitating automated retrievals of open data and post-processing with open source R functions. It allows the user to 1) rapidly assemble a quantitative, comprehensive assessment of flows thorough an urban area, and 2) easily change the spatial and temporal boundaries of analysis. We use CityWaterBalance to evaluate the water system in the Chicago metropolitan area on a monthly basis for water years 2001-2010. Results are used to consider 1) impacts of management decisions aimed at reducing stormwater and combined sewer overflows and 2) the significance of future changes in precipitation.

Estimating summer nutrient concentrations in Northeastern lakes from SPARROW load predictions and modeled lake depth and volume.

Global nutrient cycles have been altered by the use of fossil fuels and fertilizers resulting in increases in nutrient loads to aquatic systems. In the United States, excess nutrients have been repeatedly reported as the primary cause of lake water quality impairments. Setting nutrient criteria that are protective of a lakes ecological condition is one common solution; however, the data required to do this are not always easily available. A useful solution for this is to combine available field data (i.e., The United States Environmental Protection Agency (USEPA) National Lake Assessment (NLA)) with average annual nutrient load models (i.e., USGS SPARROW model) to estimate summer concentrations across a large number of lakes. In this paper we use this combined approach and compare the observed total nitrogen (TN) and total phosphorus (TN) concentrations in Northeastern lakes from the 2007 National Lake Assessment to those predicted by the Northeast SPARROW model. We successfully adjusted the SPARROW predictions to the NLA observations with the use of Vollenweider equations, simple input-output models that predict nutrient concentrations in lakes based on nutrient loads and hydraulic residence time. This allows us to better predict summer concentrations of TN and TP in Northeastern lakes and ponds. On average we improved our predicted concentrations of TN and TP with Vollenweider models by 18.7% for nitrogen and 19.0% for phosphorus. These improved predictions are being used in other studies to model ecosystem services (e.g., aesthetics) and dis-services (e.g. cyanobacterial blooms) for ~18,000 lakes in the Northeastern United States.

Ecosystem services altered by human changes in the nitrogen cycle: a new perspective for US decision making.

Human alteration of the nitrogen (N) cycle has produced benefits for health and well-being, but excess N has altered many ecosystems and degraded air and water quality. US regulations mandate protection of the environment in terms that directly connect to ecosystem services. Here, we review the science quantifying effects of N on key ecosystem services, and compare the costs of N-related impacts or mitigation using the metric of cost per unit of N. Damage costs to the provision of clean air, reflected by impaired human respiratory health, are well characterized and fairly high (e.g. costs of ozone and particulate damages of $28 per kg NO(x)-N). Damage to services associated with productivity, biodiversity, recreation and clean water are less certain and although generally lower, these costs are quite variable (<$2.2-56 per kg N). In the current Chesapeake Bay restoration effort, for example, the collection of available damage costs clearly exceeds the projected abatement costs to reduce N loads to the Bay ($8-15 per kg N). Explicit consideration and accounting of effects on multiple ecosystem services provides decision-makers an integrated view of N sources, damages and abatement costs to address the significant challenges associated with reducing N pollution.

Manufacture of measles viruses.

Measles viruses have shown potent oncolytic activity as a therapeutic against a variety of human cancers in animal models and are currently being tested in clinical trials in patients. In contrast to using measles virus as a vaccine, oncolytic activity depends on high concentrations of infectious virus. For use in humans, the high-titer measles virus preparations must also be purified to remove significant levels of cellular proteins and nucleic acid resulting from the cytolytic products of measles virus replication and release. Pleomorphic measles virus must be treated as >1-µm particles that are extremely shear sensitive to maximize recoveries and retain infectivity. Therefore, to maximize the recovery of sterile, high titer infectious measles viruses, the entire production and purification process must be done using gentle conditions and aseptic processing. Here we describe a procedure applicable to the production of small (a few liters) to large (50-60 L) batches of measles virus amplified in Vero cells adapted to serum-free growth. Cell culture supernatant containing the measles virus is clarified by filtration to remove intact Vero cells and other debris, and then treated with Benzonase(®) in the presence of magnesium chloride to digest contaminating nucleic acid. The measles virus in the treated cell culture supernatant is then concentrated and purified using tangential flow filtration (TFF) and diafiltration. The concentrated and diafiltered measles virus is passed through a final clarifying filter prior to final vialing and storage at <-65°C. An infectivity assay to quantify infectious measles virus concentration based on the TCID(50) method is also described. This procedure can be readily adapted to the production and purification of measles viruses using good manufacturing practices (GMP).

A process for comparing and interpreting differences in two benthic indices in New York Harbor.

Often when various estuarine benthic indices disagree in their assessments of benthic condition, they are reflecting different aspects of benthic condition. We describe a process to screen indices for associations and, after identifying candidate metrics, evaluate metrics individually against the indices. We utilize radar plots as a multi-metric visualization tool, and conditional probability plots and receiver operating characteristic curves to evaluate associations seen in the plots. We investigated differences in two indices, the US EPA Environmental Monitoring and Assessment Program's benthic index for the Virginian Province and the New York Harbor benthic index of biotic integrity using data collected in New York Harbor and evaluated overall agreement of the indices and associations between each index and measures of habitat and sediment contamination. The indices agreed in approximately 78% of the cases. The New York Harbor benthic index of biotic integrity showed stronger associations with sediment metal contamination and grain size.

CProb: a computational tool for conducting conditional probability analysis.

Conditional probability is the probability of observing one event given that another event has occurred. In an environmental context, conditional probability helps to assess the association between an environmental contaminant (i.e., the stressor) and the ecological condition of a resource (i.e., the response). These analyses, when combined with controlled experiments and other methodologies, show great promise in evaluating ecological conditions from observational data and in defining water quality and other environmental criteria. Current applications of conditional probability analysis (CPA) are largely done via scripts or cumbersome spreadsheet routines, which may prove daunting to end-users and do not provide access to the underlying scripts. Combining spreadsheets with scripts eases computation through a familiar interface (i.e., Microsoft Excel) and creates a transparent process through full accessibility to the scripts. With this in mind, we developed a software application, CProb, as an Add-in for Microsoft Excel with R, R(D)com Server, and Visual Basic for Applications. CProb calculates and plots scatterplots, empirical cumulative distribution functions, and conditional probability. In this short communication, we describe CPA, our motivation for developing a CPA tool, and our implementation of CPA as a Microsoft Excel Add-in. Further, we illustrate the use of our software with two examples: a water quality example and a landscape example. CProb is freely available for download at http://www.epa.gov/emap/nca/html/regions/cprob.

Predicting estuarine sediment metal concentrations and inferred ecological conditions: an information theoretic approach.

Empirically derived relationships associating sediment metal concentrations with degraded ecological conditions provide important information to assess estuarine condition. Resources limit the number, magnitude, and frequency of monitoring activities to acquire these data. Models that use available information and simple statistical relationships to predict sediment metal concentrations could provide an important tool for environmental assessment. We developed 45 predictive models for the total concentrations of copper, lead, mercury, and cadmium in estuarine sediments along the Southern New England and Mid-Atlantic regions of the United States. Using information theoretic model-averaging approaches, we found total developed land and percent silt/clay of estuarine sediment were the most important variables for predicting the presence of all four metals. Estuary area, river flow, tidal range, and total agricultural land varied in their importance. The model-averaged predictions explained 78.4, 70.5, 56.4, and 50.3% of the variation for copper, lead, mercury, and cadmium, respectively. Overall prediction accuracies of selected sediment benchmark values (i.e., effects ranges) were 83.9, 84.8, 78.6, and 92.0% for copper, lead, mercury, and cadmium, respectively. Our results further support the generally accepted conclusion that sediment metal concentrations are best described by the physical characteristics of the estuarine sediment and the total amount of urban land in the contributing watershed. We demonstrated that broad-scale predictive models built from existing monitoring data with information theoretic model-averaging approaches provide valuable predictions of estuarine sediment metal concentrations and show promise for future environmental modeling efforts in other regions.

Lixazinone stimulates mitogenesis of Madin-Darby canine kidney cells.

Polycystic kidney diseases (PKD) are characterized by excessive proliferation of renal tubular epithelial cells, development of fluid-filled cysts, and progressive renal insufficiency. cAMP inhibits proliferation of normal renal tubular epithelial cells but stimulates proliferation of renal tubular epithelial cells derived from patients with PKD. Madin-Darby canine kidney (MDCK) epithelial cells, which are widely used as an in vitro model of cystogenesis, also proliferate in response to cAMP. Intracellular cAMP levels are tightly regulated by phosphodiesterases (PDE). Isoform-specific PDE inhibitors have been developed as therapeutic agents to regulate signaling pathways directed by cAMP. In other renal cell types, we have previously demonstrated that cAMP is hydrolyzed by PDE3 and PDE4, but only PDE3 inhibitors suppress proliferation by inhibiting Raf-1 activity (Cheng J, Thompson MA, Walker HJ, Gray CE, Diaz Encarnacion MM, Warner GM, Grande JP. Am J Physiol Renal Physiol 287:F940-F953, 2004.) A potential role for PDE isoform(s) in cAMP-mediated proliferation of MDCK cells has not previously been established. Similar to what we have previously found in several other renal cell types, cAMP hydrolysis in MDCK cells is directed primarily by PDE4 (85% of total activity) and PDE3 (15% of total activity). PDE4 inhibitors are more effective than PDE3 inhibitors in increasing intracellular cAMP levels in MDCK cells. However, only PDE3 inhibitors, and not PDE4 inhibitors, stimulate mitogenesis of MDCK cells. PDE3 but not PDE4 inhibitors activate B-Raf but not Raf-1, as assessed by an in vitro kinase assay. PDE3 but not PDE4 inhibitors activate the ERK pathway and activate cyclins D and E, as assessed by histone H1 kinase assay. We conclude that mitogenesis of MDCK cells is regulated by a functionally compartmentalized intracellular cAMP pool directed by PDE3. Pharmacologic agents that stimulate PDE3 activity may provide the basis for new therapies directed toward reducing cystogenesis in patients with PKD.

Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4.

Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.

Differential effects of low-dose docosahexaenoic acid and eicosapentaenoic acid on the regulation of mitogenic signaling pathways in mesangial cells.

Although dietary fish oil supplementation has been used to prevent the progression of kidney disease in patients with IgA nephropathy, relatively few studies provide a mechanistic rationale for its use. Using an antithymocyte (ATS) model of mesangial proliferative glomerulonephritis, we recently demonstrated that fish oil inhibits mesangial cell (MC) activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. We therefore sought to define potential mechanisms underlying the antiproliferative effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the predominant omega-3 polyunsaturated fatty acids found in fish oil, in cultured MC. DHA and EPA were administered to MC as bovine serum albumin fatty-acid complexes. Low-dose (10-50 micromol/L) DHA, but not EPA, inhibited basal and epidermal growth factor (EGF)-stimulated [(3)H]-thymidine incorporation in MCs. At higher doses (100 micromol/L), EPA and DHA were equally effective in suppressing basal and EGF-stimulated MC mitogenesis. Low-dose DHA, but not EPA, decreased ERK activation by 30% (P <.01), as assessed with Western-blot analysis using phosphospecific antibodies. JNK activity was increased by low-dose DHA but not by EPA. p38 activity was not significantly altered by DHA or EPA. Cyclin E activity, as assessed with a histone H1 kinase assay, was inhibited by low-dose DHA but not by EPA. DHA increased expression of the cell cycle inhibitor p21 but not p27; EPA had no effect on p21 or p27. We propose that the differential effect of low-dose DHA vs EPA in suppressing MC mitogenesis is related to down-regulation of ERK and cyclin E activity and to induction of p21.

TGF-beta1 is an autocrine mediator of renal tubular epithelial cell growth and collagen IV production.

Recent studies in cultured cells have provided evidence that a variety of pathobiologic stimuli, including high glucose, angiotensin II, and thromboxane A(2), trigger a signaling pathway leading to autocrine induction of TGF-beta1. TGF-beta1 production through this pathway may profoundly affect cell growth, matrix synthesis, and response to injury. This study examines the role of autocrine versus exogenously added TGF-beta1 in cellular proliferation and collagen IV production, critical targets of TGF-beta1 signaling, using renal cells derived from TGF-beta1 knockout (KO) animals or wild-type (WT) controls. Growth of WT and KO cells was assessed by cell counting and [(3)H]thymidine uptake. Basal and TGF-beta1-stimulated collagen production was assessed by Northern and Western blotting; transcriptional activity of the alpha1(IV) collagen gene was assessed by transient transfection analysis. KO cells grew at a faster rate than WT cells carefully matched for plating density and passage number. This increased growth rate was paralleled by increases in [(3)H]thymidine uptake. KO cells expressed lower levels of the cell cycle inhibitors p21 and p27 than WT cells. KO cells failed to express TGF-beta1, as expected. Basal TGF-beta3 mRNA levels were higher in KO cells than in WT cells. WT cells expressed higher basal levels of TGF-beta2 mRNA than KO cells. Basal alpha1(IV) and alpha2(IV) collagen mRNA and protein expression were significantly lower in KO cells than WT cells. Administration of exogenous TGF-beta1 induced collagen IV production in both KO and WT cells. Although basal transcriptional activity of an alpha1(IV) collagen-CAT construct was lower in KO cells than WT cells, administration of exogenous TGF-beta1 was associated with significant increases in transcriptional activity of this construct in both KO and WT cells. These studies provide evidence that autocrine production of TGF-beta1 may play a critical role in regulation of growth and basal collagen IV production by renal tubular epithelial cells.