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Organomodified nanoclays (ONCs: i.e., smectite clays with different organic coatings), act as nanofillers in applications ranging from automotive, to aerospace, and biomedical implementations. Because of their large utilization, ONC industry is projected to be a U.S. $3.3 billion industry by 2023. However, emerging studies showed that ONCs and ONC-nanocomposites could harbor health risks upon pulmonary exposure and along their life cycle, namely during synthesis, handling, use, manipulation, and disposal. Specifically, the potential for exposure and adverse effects on human health primarily includes release of dry particulate during handling, manipulation, release during use, and release during the end-of-life disposal or recycling scenarios. Compared to other ENMs, however, little information exists describing which physicochemical properties contribute to their health risk. My M.S. work at West Virginia University in Prof. Dinu's group aimed to evaluate acute toxicity of a library of ONCs prior to and after simulated disposal by incineration. The analysis conducted high content screening and real-time electric cell impedance sensing on bronchial epithelial cell monolayers for coupled high-throughput in vitro assessment strategies aimed to evaluate acute toxicity of a library of ONCs prior to and after simulated disposal by incinceration. Coating-, incineration status-, and time-dependent effects were considered to determine changes in the pulmonary airway epithelial monolayer integrity, cell transepithelial resistance, apoptosis, and cell metabolism respectively. Results showed that after exposure to each particle at its IC50, pristine nanoclay displayed acute loss of monolayer coverage, resistance, and metabolism, coupled with increased number of apoptotic cells. Conversely, three different ONCs of prevalent use displayed little loss of monolayer integrity but exhibited differential coating-depended increased apoptosis and up to 40-45% initial reduction in cell metabolism. Moreover, incinerated byproducts of ONCs exhibited significant loss of monolayer coverage and integrity, increased necrosis, and little evidence of monolayer re-establishment. These findings indicate that characteristics of organic coating type largely determine mechanism of cytotoxicity and the ability of the monolayer to recover. Use of high content screening, coupled with traditional in vitro assays prove to serve as a rapid pulmonary toxicity assessment tool to thus help define prevention by targeted physicochemical material properties design strategies. The above contributions to understanding deleterious effect of nanomaterials on human model systems is meritorious and transformative and allows for creating new tools for toxicological pathways assessment to be considered for improved prevention by design strategies and enhanced safety practices. The above contributions has been included in a co-first author publication, namely: Stueckle, T.*, White, A.*, Wager, A., Gupta, R., Rojanasakul, Y., Dinu, C.Z., "Impacts of Organomodified Nanoclays and their Incincerated Byproducts on Bronchial Cell Monolayer Integrity", Chemical Research in Toxicology (2019) 32, 12, 2445-2458 (*Authors contributed equally).

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum drug resistance and can decrease the bioavailability of many important drugs such as anti-cancer agents, antibiotics, antivirals, antidepressants and antiinflammatory drugs. Calcein-AM, a fluorescent reporter commonly used for studying compound interactions with MRP1 was recently used in the development of a high content imaging-based assay by our group. This assay was robust and had better sensitivity than fluorescent plate readouts. The assay identified 12 MRP1 inhibitors after screening an anticancer library of 386 compounds. Due to the multiple distinct substrate binding sites of MRP1, we sought to use different fluorescent probes to identify substrate selective inhibitors which were likely missed by the calcein-AM screening. The high content imaging-based uptake assay was modified using doxorubicin (anticancer drug) and CRO- 9 (dye) as fluorescent reporters which vary in structure and function. The doxorubicin assay, after screening the same 386 compound library identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. The CRO-9 assay identified a total of 50 MRP1 inhibitors including 19 additional inhibitors that have never been reported as inhibitors of MRP1. These 50 MRP1 inhibitors included 10 out of 12 hits identified using calcein-AM and 27 out of 28 inhibitors discovered through the doxorubicin assay. MRP1 inhibition was confirmed using flow cytometry, confocal microscopy and membrane-based transport assays. Selected drugs were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against various substrates. From the doxorubicin screening, mifepristone and doramapimod were the most effective in reversing MRP1 mediated resistance whiles celecoxib exhibited selective MRP1 inhibition. From the hits identified through the CRO- 9-based screening, LY2603618 and ZSTK474 were the most effective in reversing MRP1 mediated resistance in H69AR cells. Together, our findings signify the effectiveness and value of doxorubicin and CRO-9 based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies.

Images of cells incubated with fluorescent small molecule probes can be used to infer where the compounds distribute within cells. Identifying the spatial pattern of compound localization within each cell is very important problem for which adequate statistical methods do not yet exist. First, we asked whether a classifier for subcellular localization categories can be developed based on a training set of manually classified cells. Due to challenges of the images such as uneven field illumination, low resolution, high noise, variation in intensity and contrast, and cell to cell variability in probe distributions, we constructed texture features for contrast quantiles conditioning on intensities, and classifying on artificial cells with same marginal distribution but different conditional distribution supported that this conditioning approach is beneficial to distinguish different localization distributions. Using these conditional features, we obtained satisfactory performance in image classification, and performed to dimension reduction and data visualization. As high content images are subject to several major forms of artifacts, we are interested in the implications of measurement errors and artifacts on our ability to draw scientifically meaningful conclusions from high content images. Specifically, we considered three forms of artifacts: saturation, blurring and additive noise. For each type of artifacts, we artificially introduced larger amount, and aimed to understand the bias by `Simulation Extrapolation' (SIMEX) method, applied to the measurement errors for pairwise centroid distances, the degree of eccentricity in the class-specific distributions, and the angles between the dominant axes of variability for different categories. Finally, we briefly considered the analysis of time-point images. Small molecule studies will be more focused. Specifically, we consider the evolving patterns of subcellular staining from the moment that a compound is introduced into the cell culture medium, to the point that steady state distribution is reached. We construct the degree to which the subcellular staining pattern is concentrated in or near the nucleus as the features of timecourse data set, and aim to determine whether different compounds accumulate in different regions at different times, as characterized in terms of their position in the cell relative to the nucleus.

Human pluripotent stem cells (hPSCs) can be derived from the inner cell mass of a developing blastocyst or through transcription factor mediated reprogramming. hPSCs can be maintained indefinitely in culture and have the capacity to differentiate into any cell type found in the body, making them powerful tools for studying development, modeling diseases, and potential regenerative medicine therapies. Before we can utilize the full potential of these cells, there are many unanswered questions remaining regarding their basic growth and survival. While hPSCs are regularly grown as compact colonies and passaged as small clumps, when dissociated to single cells they survive poorly and undergo apoptosis rapidly after dissociation. This poor survival as single cells impairs the use of hPSCs in common techniques that require the isolation of specific clonal populations, including genetic manipulation. Towards this end, we have taken several approaches, including candidate targeting and large scale screening, to identify modifiers of survival in hPSCs. One such small molecule that significantly improved the survival of dissociated hPSCs was the Rho kinase (ROCK) inhibitor HA-1077. Using shRNA we confirmed that inhibiting ROCK activity in hPSCs improves their survival by inhibiting dissociation-induced apoptosis. We also explored the role of cell-cell junctions between hPSCs in regulating survival. We found that blocking the activity of E-cadherin in dissociated cells reduces their survival and plating efficiency, even when treated with a ROCK inhibitor. Furthermore, we identified multiple junction proteins that are disrupted after dissociation of hPSC colonies, representing novel targets with potential to regulate survival of hPSCs. Finally, we developed improved high throughput assays for screening large libraries of small molecules to search for novel regulators of hPSC survival. We identified several novel small molecules, and validated that most function through inhibition of ROCK, suggesting that ROCK activity is a key modulator of survival in hPSCs. The data reported here represent a comprehensive evaluation of how survival is regulated in hPSCs. Further this work is one of the first to generate and compare large scale screening platforms for hPSC survival. With minor modifications, these screening platforms can be adapted to examine other aspects of hPSC fate such as self-renewal and differentiation. Improving our ability to grow, maintain, and manipulate hPSCs will enhance their use as a model for human disease and development and a source of future cell-based regenerative therapies.