Inducing Deep Sweeps and Vortex Ejections on Patterned Membrane Surfaces to Mitigate Surface Fouling.

Patterned membrane surfaces offer a hydrodynamic approach to mitigating concentration polarization and subsequent surface fouling. However, when subjected to steady crossflow conditions, surface patterns promote particle accumulation in the recirculation zones of cavity-like spaces. In order to resolve this issue, we numerically subject a two-dimensional, patterned membrane surface to a rapidly pulsed crossflow. When combined with cavity-like spaces, such as the valleys of membrane surface patterns, a rapidly pulsed flow generates mixing mechanisms (i.e., the deep sweep and the vortex ejection) and disrupts recirculation zones. In only four pulses, we demonstrate the ability of these mechanisms to remove over half of the particles trapped in recirculation zones via massless particle tracking studies (i.e., numerical integration of the simulated velocity field). The results of this work suggest that when combined with a rapidly pulsed inlet flow, patterned membrane surfaces can not only alleviate concentration polarization and the surface fouling that follows but also reduce the need for traditional cleaning methods that require operational downtime and often involve the use of abrasive chemical agents.

A hands-free stool sampling system for monitoring intestinal health and disease.

Analysis of stool offers simple, non-invasive monitoring for many gastrointestinal (GI) diseases and access to the gut microbiome, however adherence to stool sampling protocols remains a major challenge because of the prevalent dislike of handling one's feces. We present a technology that enables individual stool specimen collection from toilet wastewater for fecal protein and molecular assay. Human stool specimens and a benchtop test platform integrated with a commercial toilet were used to demonstrate reliable specimen collection over a wide range of stool consistencies by solid/liquid separation followed by spray-erosion. The obtained fecal suspensions were used to perform occult blood tests for GI cancer screening and for microbiome 16S rRNA analysis. Using occult blood home test kits, we found overall 90% agreement with standard sampling, 96% sensitivity and 86% specificity. Microbiome analysis revealed no significant difference in within-sample species diversity compared to standard sampling and specimen cross-contamination was below the detection limit of the assay. Furthermore, we report on the use of an analogue turbidity sensor to assess in real time loose stools for tracking of diarrhea. Implementation of this technology in residential settings will improve the quality of GI healthcare by facilitating increased adherence to routine stool monitoring.

Intracerebral hemorrhage and thrombin-induced alterations in cerebral microvessel matrix.

Four phase III clinical trials of oral direct factor Xa or thrombin inhibitors demonstrated significantly lower intracranial hemorrhage compared to warfarin in patients with nonvalvular-atrial fibrillation. This is counter-intuitive to the principle that inhibiting thrombosis should increase hemorrhagic risk. We tested the novel hypothesis that anti-thrombin activity decreases the risk of intracerebral hemorrhage by directly inhibiting thrombin-mediated degradation of cerebral microvessel basal lamina matrix, responsible for preventing hemorrhage. Collagen IV, laminin, and perlecan each contain one or more copies of the unique α-thrombin cleavage site consensus sequence. In blinded controlled experiments, α-thrombin significantly degraded each matrix protein in vitro and in vivo in a concentration-dependent fashion. In vivo stereotaxic injection of α-thrombin significantly increased permeability, local IgG extravasation, and hemoglobin (Hgb) deposition together with microvessel matrix degradation in a mouse model. In all formats the direct anti-thrombin dabigatran completely inhibited matrix degradation by α-thrombin. Fourteen-day oral exposure to dabigatran etexilate-containing chow completely inhibited matrix degradation, the permeability to large molecules, and cerebral hemorrhage associated with α-thrombin. These experiments demonstrate that thrombin can degrade microvessel matrix, leading to hemorrhage, and that inhibition of microvessel matrix degradation by α-thrombin decreases cerebral hemorrhage. Implications for focal ischemia and other conditions are discussed.

Laboratory Demonstration and Preliminary Techno-Economic Analysis of an Onsite Wastewater Treatment System.

Providing safe and reliable sanitation services to the billions of people currently lacking them will require a multiplicity of approaches. Improving onsite wastewater treatment to standards enabling water reuse would reduce the need to transport waste and fresh water over long distances. Here, we describe a compact, automated system designed to treat the liquid fraction of blackwater for onsite water reuse that combines cross-flow ultrafiltration, activated carbon, and electrochemical oxidation. In laboratory testing, the system consistently produces effluent with 6 ≤ pH ≤ 9, total suspended solids (TSS) < 30 mg L-1, and chemical oxygen demand (COD) < 150 mg L-1. These effluent parameters were achieved across a wide range of values for influent TSS (61-820 mg L-1) and COD (384-1505 mg L-1), demonstrating a robust system for treating wastewater of varying strengths. A preliminary techno-economic analysis (TEA) was conducted to elucidate primary cost drivers and prioritize research and development pathways toward commercial feasibility. The ultrafiltration system is the primary cost driver, contributing to >50% of both the energy and maintenance costs. Several scenario parameters showed an outsized impact on costs relative to technology parameters. Specific technological improvements for future prototype development are discussed.

Resolving the relative contributions of cistern and pour flushing to toilet water usage: Measurements from urban test sites in India.

A challenge in water reuse for toilet flushing in India and other Asian countries derives from pour flushing practices. It is a common assumption that the amount of pour flushed water used for personal cleansing is small in comparison to the cistern flush volume, however there is a knowledge gap regarding the actual contribution of each water source to the blackwater amount. In this study, digital water meters were used to measure the fraction of water from personal wash tap relative to cistern water that is used for toilet flushing. High temporal resolution measurements were carried in three different urban sites in the city of Coimbatore in the southern Indian state of Tamil Nadu where onsite sanitation treatment prototypes that may provide reclaimed water for cistern flushing are being tested. Data collected over periods of up to 2 months show that the contribution of the cistern flush to the total blackwater volume is low (14-40%). These data highlight an important factor to inform interventions designed around water reuse for flushing in world geographies where personal toilet cleansing by water is the common practice.

Field testing of a household-scale onsite blackwater treatment system in Coimbatore, India.

4.2 billion people live without access to safely managed sanitation services. This report describes the field testing of an onsite prototype system designed to treat blackwater from a single flush toilet and reuse of the treated effluent for flushing. The system passes wastewater through a solid-liquid separator followed by settling tanks and granular activated carbon columns into an electrochemical reactor that oxidizes chloride salts from urine to generate chlorine to remove pathogens. The objectives of the study were to verify the functionality of the system (previously demonstrated in the laboratory) under realistic use conditions, to identify maintenance requirements, and to make a preliminary assessment of the system's user acceptability. The prototype was installed in a women's workplace and residential toilet block in Coimbatore, India, and tested over a period of 10 months. The treated water met stringent disinfection threshold for both E. coli and helminth eggs and produced a clear, colorless effluent that met or nearly met local and international discharge standards for non-sewered sanitation systems. The effluent had an average chemical oxygen demand of 81 mg/L, total suspended solids of 11 mg/L, and reduction of total nitrogen by 65%. These tests determined the recommended service lifetimes and maintenance intervals for key system components including the electrochemical cell, granular activated carbon columns, and solid-liquid separator. User feedback regarding the use of treated blackwater as flush water was positive. These findings will inform the design and implementation of next-generation systems currently under development.

Field testing of a household-scale onsite blackwater treatment system in South Africa.

Innovations that enable cost-effective and resource-conserving treatment of human waste are required for the 4.2 billion people in the world who currently lack safe and reliable sanitation services. Onsite treatment and reuse of blackwater is one strategy towards this end, greatly reducing the need to transport wastewater over long distances either via sewers or trucks. Here, we report on the field testing of a prototype onsite blackwater treatment system conducted over a period of 8 months. The system was connected to a women's toilet in a public communal ablution block located in an informal settlement near Durban, South Africa. Liquid waste was treated by separation and diversion of large solids, settling of suspended solids, and filtration through activated carbon prior to disinfection by electrochemical oxidation. System performance was monitored daily by measurement of chemical and physical water quality parameters onsite and confirmed by periodic detailed analysis of chemical and biological parameters at an offsite lab. Daily monitoring of system performance indicated that the effluent had minimal color and turbidity (maximum 90 Pt/Co units and 6.48 NTU, respectively), and consistent evolution of chlorine as blackwater passed through the system. Weekly offsite analysis confirmed that the system consistently inactivated pathogens (E. coli and coliforms) and reduced chemical oxygen demand and total suspended solids to meet ISO 30500 category B standards. Significant reductions in total nitrogen load were also observed, though these reductions often fell short of the 70% reduction required by ISO 30500. No significant reduction in total phosphorus was observed. Maintenance requirements were identified, and the resilience of the system to restart following a prolonged shutdown was demonstrated, but significant improvements are required in the design of the solid/liquid separation mechanism for application of this system in a wiping culture.

Remediation of suspended solids and turbidity by improved settling tank design in a small-scale, free-standing toilet system using recycled blackwater.

Our research is focused on the development of decentralized waste water treatment technologies enabling onsite water reuse. Accumulation of solids with recycling of treated blackwater increases the energy required for disinfection with an electrochemical process. We hypothesized that improving the preprocess settling of blackwater by increasing the tortuosity of the liquid flow path would reduce this energy demand by reducing particle-associated chemical oxygen demand (COD). This approach successfully reduced the total suspended solids and turbidity in the process liquid accumulated per user-day equivalent. A modest reduction in the apparent steady-state accumulation of COD was also observed, likely because of the retention of COD associated with larger particles in the settling tanks. Interestingly, these improvements did not improve the energy efficiency of the electrochemical disinfection process, as predicted. These observations suggest that improving the energy efficiency of electrochemical disinfection will require remediation of dissolved COD.

Non-biological methods for phosphorus and nitrogen removal from wastewater: A gap analysis of reinvented-toilet technologies with respect to ISO 30500.

The aims of the Reinvent the Toilet Challenge (RTTC) include creation of an off-the-grid sanitation system with operating costs of less than US$0.05 per user per day. Because of the small scale at which many reinvented toilets (RT) are intended to operate, non-biological treatment has been generally favored. The RTTC has already instigated notable technological advances in non-sewered sanitation systems (NSSS). However, increasingly stringent liquid effluent standards for N and P could limit the deployment of current RT in real-world scenarios, despite the urgent need for these systems. The newly adopted ISO 30500 standards for water reuse in NSSS dictate minimal use of chemical/biological additives, while at the same time requiring a 70% and 80% reduction in total nitrogen and phosphorus, respectively. This document provides a brief overview of the mature and emerging technologies for N and P (specifically ammonia/ammonium and orthophosphate) removal from wastewater. At present, the dearth of nutrient removal methods proven to be effective at small scales is a significant barrier to meeting ISO 30500 standards. Closing the gap between RTs and ISO 30500 will require significant investments in basic R&D of emerging technologies for non-biological N and P remediation and/or increased reliance on biological processes. Adaptation of existing nutrient-removal technologies to small-scale NSSS is a viable option that merits additional investigation.

A granular activated carbon/electrochemical hybrid system for onsite treatment and reuse of blackwater.

Over 1/3 of the global population lacks access to improved sanitation, leading to disease, death, and impaired economic development. Our group is working to develop rapidly deployable, cost-effective, and sustainable solutions to this global problem that do not require significant investments in infrastructure. Previously, we demonstrated the feasibility of a toilet system that recycles blackwater for onsite reuse as flush water, in which the blackwater is electrochemically treated to remove pathogens due to fecal contamination. However, this process requires considerable energy (48-93 kJ/L) to achieve complete disinfection of the process liquid, and the disinfected liquid retains color and chemical oxygen demand (COD) in excess of local discharge standards, negatively impacting user acceptability. Granular activated carbon (GAC) efficiently reduces COD in concentrated wastewaters. We hypothesized that reduction of COD with GAC prior to electrochemical treatment would both improve disinfection energy efficiency and user acceptability of the treated liquid. Here we describe the development and testing of a hybrid system that combines these technologies and demonstrate its ability to achieve full disinfection with improved energy efficiency and liquid quality more suitable for onsite reuse and/or discharge.

Three-dimensional (3D) tetra-culture brain on chip platform for organophosphate toxicity screening.

Organophosphate-based compounds (OPs) represent a significant threat to warfighters (nerve agents) and civilian populations (pesticides). There is a pressing need to develop in vitro brain models that correlate to the in vivo brain to rapidly study OPs for neurotoxicity. Here we report on a microfluidic-based three-dimensional, four-cell tissue construct consisting of 1) a blood-brain barrier that has dynamic flow and membrane-free culture of the endothelial layer, and 2) an extracellular matrix (ECM)-embedded tissue construct with neuroblastoma, microglia, and astrocytes. We demonstrated this platform's utility by measuring OP effects on barrier integrity, acetylcholinesterase (AChE) inhibition, viability and residual OP concentration with four model OPs. The results show that the OPs penetrate the blood brain barrier (BBB) and rapidly inhibit AChE activity, and that in vitro toxicity was correlated with available in vivo data. This paper demonstrates the potential utility of a membrane-free tetra-cultured brain on chip that can be scaled to high throughput as a cost-effective alternative method to animal testing.

ß1-integrin-matrix interactions modulate cerebral microvessel endothelial cell tight junction expression and permeability.

Acutely following focal cerebral ischemia disruption of the microvessel blood-brain barrier allows transit of plasma proteins into the neuropil as edema formation that coincides with loss of microvessel endothelial ß1-integrins. We extend previous findings to show that interference with endothelial ß1-integrin-matrix adhesion by the monoclonal IgM Ha2/5 increases the permeability of primary cerebral microvascular endothelial cell monolayers through reorganization of claudin-5, occludin, and zonula occludens-1 (ZO-1) from inter-endothelial borders. Interference with ß1-integrin-matrix adhesion initiates F-actin conformational changes that coincide with claudin-5 redistribution. ß1-integrin-matrix interference simultaneously increases phosphorylation of myosin light chain (MLC), while inhibition of MLC kinase (MLCK) and Rho kinase (ROCK) abolishes the Ha2/5-dependent increased endothelial permeability by 6 h after ß1-integrin-matrix interference. These observations are supported by concordant observations in the cortex of a high-quality murine conditional ß1-integrin deletion construct. Together they support the hypothesis that detachment of ß1-integrins from abluminal matrix ligands increases vascular endothelial permeability through reorganization of tight junction (TJ) proteins via altered F-actin conformation, and indicate that the ß1-integrin-MLC signaling pathway is engaged when ß1-integrin detachment occurs. These findings provide a novel approach to the research and treatment of cerebral disorders where the breakdown of the blood-brain barrier accounts for their progression and complication.

Improving energy efficiency of electrochemical blackwater disinfection through sequential reduction of suspended solids and chemical oxygen demand.

Onsite reuse of blackwater requires removal of considerable amounts of suspended solids and organic material in addition to inactivation of pathogens. Previously, we showed that electrochemical treatment could be used for effective pathogen inactivation in blackwater, but was inadequate to remove solids and organics to emerging industry standards. Further, we found that as solids and organics accumulate with repeated recycling, electrochemical treatment becomes less energetically sustainable. Here, we describe a pilot study in which concentrated blackwater is pretreated with ultrafiltration and granular activated carbon prior to electrochemical disinfection, and show that this combination of treatments removes 75-99% of chemical oxygen demand, 92-100% of total suspended solids, and improves the energy efficiency of electrochemical blackwater treatment by an order of magnitude.

Electrochemical disinfection of repeatedly recycled blackwater in a free-standing, additive-free toilet.

Decentralized, energy-efficient waste water treatment technologies enabling water reuse are needed to sustainably address sanitation needs in water- and energy-scarce environments. Here, we describe the effects of repeated recycling of disinfected blackwater (as flush liquid) on the energy required to achieve full disinfection with an electrochemical process in a prototype toilet system. The recycled liquid rapidly reached a steady state with total solids reliably ranging between 0.50 and 0.65% and conductivity between 20 and 23 mS/cm through many flush cycles over 15 weeks. The increase in accumulated solids was associated with increased energy demand and wide variation in the free chlorine contact time required to achieve complete disinfection. Further studies on the system at steady state revealed that running at higher voltage modestly improves energy efficiency, and established running parameters that reliably achieve disinfection at fixed run times. These results will guide prototype testing in the field.

Enhanced H2O2 Production at Reductive Potentials from Oxidized Boron-Doped Ultrananocrystalline Diamond Electrodes.

This work investigates the surface chemistry of H2O2 generation on a boron-doped ultrananocrystalline diamond (BD-UNCD) electrode. It is motivated by the need to efficiently disinfect liquid waste in resource constrained environments with limited electrical power. X-ray photoelectron spectroscopy was used to identify functional groups on the BD-UNCD electrode surfaces while the electrochemical potentials of generation for these functional groups were determined via cyclic voltammetry, chronocoulometry, and chronoamperometry. A colorimetric technique was employed to determine the concentration and current efficiency of H2O2 produced at different potentials. Results showed that preanodization of an as-grown BD-UNCD electrode can enhance the production of H2O2 in a strong acidic environment (pH 0.5) at reductive potentials. It is proposed that the electrogeneration of functional groups at oxidative potentials during preanodization allows for an increased current density during the successive electrolysis at reductive potentials that correlates to an enhanced production of H2O2. Through potential cycling methods, and by optimizing the applied potentials and duty cycle, the functional groups can be stabilized allowing continuous production of H2O2 more efficiently compared to static potential methods.

Systems biology for organotypic cell cultures.

Translating in vitro biological data into actionable information related to human health holds the potential to improve disease treatment and risk assessment of chemical exposures. While genomics has identified regulatory pathways at the cellular level, translation to the organism level requires a multiscale approach accounting for intra-cellular regulation, inter-cellular interaction, and tissue/organ-level effects. Tissue-level effects can now be probed in vitro thanks to recently developed systems of three-dimensional (3D), multicellular, "organotypic" cell cultures, which mimic functional responses of living tissue. However, there remains a knowledge gap regarding interactions across different biological scales, complicating accurate prediction of health outcomes from molecular/genomic data and tissue responses. Systems biology aims at mathematical modeling of complex, non-linear biological systems. We propose to apply a systems biology approach to achieve a computational representation of tissue-level physiological responses by integrating empirical data derived from organotypic culture systems with computational models of intracellular pathways to better predict human responses. Successful implementation of this integrated approach will provide a powerful tool for faster, more accurate and cost-effective screening of potential toxicants and therapeutics. On September 11, 2015, an interdisciplinary group of scientists, engineers, and clinicians gathered for a workshop in Research Triangle Park, North Carolina, to discuss this ambitious goal. Participants represented laboratory-based and computational modeling approaches to pharmacology and toxicology, as well as the pharmaceutical industry, government, non-profits, and academia. Discussions focused on identifying critical system perturbations to model, the computational tools required, and the experimental approaches best suited to generating key data.

An optically transparent membrane supports shear stress studies in a three-dimensional microfluidic neurovascular unit model.

We report a microfluidic blood-brain barrier model that enables both physiological shear stress and optical transparency throughout the device. Brain endothelial cells grown in an optically transparent membrane-integrated microfluidic device were able to withstand physiological fluid shear stress using a hydrophilized polytetrafluoroethylene nanoporous membrane instead of the more commonly used polyester membrane. A functional three-dimensional microfluidic co-culture model of the neurovascular unit is presented that incorporates astrocytes in a 3D hydrogel and enables physiological shear stress on the membrane-supported endothelial cell layer.

Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor ß1.

Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)ß1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFß1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit.

Hemostasis and alterations of the central nervous system.

Modulation of coagulation has been successfully applied to ischemic disorders of the central nervous system (CNS). Some components of the coagulation system have been identified in the CNS, yet with limited exception their functions have not been clearly defined. Little is known about how events within the cerebral tissues affect hemostasis. Nonetheless, the interaction between cerebral cells and vascular hemostasis and the possibility that endogenous coagulation factors can participate in functions within the neurovascular unit provide intriguing possibilities for deeper insight into CNS functions and the potential for treatment of CNS injuries. Here, we consider the expression of coagulation factors in the CNS, the coagulopathy associated with focal cerebral ischemia (and its relationship to hemorrhagic transformation), the use of recombinant tissue plasminogen activator (rt-PA) in ischemic stroke and its study in animal models, the impact of rt-PA on neuron and CNS structure and function, and matrix protease generation and matrix degradation and hemostasis. Interwoven among these topics is evidence for interactions of coagulation factors with and within the CNS. How activation of hemostasis occurs in the cerebral tissues and how the brain responds are difficult questions that offer many research possibilities.

Disruption of dystroglycan-laminin interactions modulates water uptake by astrocytes.

Cerebral edema is a serious complication of ischemic brain injury. Cerebral edema includes accumulation of extracellular fluid due to leakage of the brain's microvessel permeability barrier, and swelling of astrocytes as they absorb water from the extracellular space. Expression of matrix adhesion receptors in brain microvessels decreases in ischemic stroke; this contributes to increased microvessel permeability and detachment of astrocytes from the extracellular matrix (ECM). Since loss of the astrocyte adhesion receptor dystroglycan has been associated with disrupted polarization of ion and water channels, we hypothesized that adhesion of astrocytes to the ECM contributes to regulation of water uptake, and that disruption of matrix adhesion impairs the ability of astrocytes to direct water transport. To test this hypothesis, the capacity of astrocytes to take up water was measured using a fluorescence self-quenching assay under both oxygen/glucose deprivation (OGD) and direct antibody-mediated blockade of α-dystroglycan. Both conditions decreased the rate of water uptake. Moreover, inhibiting proteolytic cleavage of dystroglycan that occurs in OGD abrogated the effect of OGD, but not direct blockade of α-dystroglycan, indicating that interfering with dystroglycan-matrix binding itself affects water uptake. Activation of extracellular signal-related kinase (ERK) by OGD was dependent on α-dystroglycan binding, and inhibition of ERK activity with U0126 abrogated the loss of water uptake following OGD. These studies demonstrate for the first time that water uptake in astrocytes is regulated by dystroglycan-dependent signaling associated with matrix adhesion. This presents a novel potential approach to the treatment of cerebral edema.