Abstract Animal studies have linked the estrogenic properties of bisphenol A (BPA) to adverse effects on the endocrine system. Because of concerns for similar effects in humans, there is a desire to replace BPA in consumer products, and a search for BPA replacements that lack endocrine-disrupting bioactivity is ongoing. We used multiple cell-based models, including an established multi-parametric, high throughput microscopy-based platform that incorporates engineered HeLa cell lines with visible ERα- or ERβ-regulated transcription loci, to discriminate the estrogen-like and androgen-like properties of previously uncharacterized substituted bisphenol derivatives and hydroquinone. As expected, BPA induced 70–80% of the estrogen-like activity via ERα and ERβ compared to E2 in the HeLa prolactin array cell line. 2,2’ BPA, Bisguaiacol F, CHDM 4-hydroxybuyl acrylate, hydroquinone, and TM modified variants of BPF showed very limited estrogen-like or androgen-like activity (< 10% of that observed with the control compounds). Interestingly, TM-BFP and CHDM 4-hydroxybuyl acrylate, but not their derivatives, demonstrated evidence of anti-estrogenic and anti-androgenic activity. Our findings indicate that Bisguaiacol F, TM-BFP-ER and TM-BPF-DGE demonstrate low potential for affecting estrogenic or androgenic endocrine activity. This suggest that the tested compounds could be suitable commercially viable alternatives to BPA.

Citation: Szafran AT, Stossi F, Mancini MG, Walker CL, Mancini MA (2017) Characterizing properties of non-estrogenic substituted bisphenol analogs using high throughput microscopy and image analysis. PLoS ONE 12(7): e0180141. https://doi.org/10.1371/journal.pone.0180141 Editor: Wei Xu, University of Wisconsin Madison, UNITED STATES Received: February 1, 2017; Accepted: June 10, 2017; Published: July 13, 2017 Copyright: © 2017 Szafran et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant summary data are within the paper. All raw and normalized data contributing to the summary figures is included in its Supporting Information files. Funding: This work was supported by NIEHS grants NIEHS R01 (1R01ES023206-01; Cheryl L. Walker, Bert W. O’Malley, M.A.M. and Mark T. Bedford) and NIEHS P30 (ES023512-01; Center of Excellence in Environmental Health, C.L.W.). Further support was provided by the Integrated Microscopy Core at Baylor College of Medicine with funding from the John S. Dunn Gulf Coast Consortium for Chemical Genomics, the Dan L. Duncan Cancer Center (NIH P30CA125123), and the NIH (HD007495, and DK56338). ATS is a K12 Scholar supported by NIH grant K12DK0083014, the multidisciplinary K12 Urologic Research (KURe) Career Development Program awarded to Dr. Dolores J. Lamb. DeepBio, Inc received funds and material support from the Valspar Corporation. Funders do not have scientific or editorial control over the design, conduct, analysis, or interpretation of the data, nor were they involved in the decision to publish or producing this manuscript. DeepBio, Inc provided support in the form of materials and consulting fees for A.T.S., F.S., and M.G.M. and provided salary for M.A.M. The specific roles of these authors are articulated in the ‘author contributions’ section. M.A.M participated in in study design, data interpretation, and final manuscript review, however, DeepBio, Inc did not have editorial control nor was involved in the decision to publish. Competing interests: M.A.M. is a founding scientist and a board member of DeepBio, an organization that provides high throughput screening and novel monoclonal antibody production services. To ensure scientific independence, A.T.S., F.S., M.G.M. performed work as external consultants to DeepBio, Inc. There are no patents, products in development or marketed products to declare by DeepBio, Inc or the authors of this manuscript. The Valspar corporation provided investigational compounds for the project but had no influence study design or data interpretation. The commercial support does not alter our adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Introduction The impact of man-made chemicals on human health and the environment is a continuous global concern. Between the thousands of compounds synthesized, chemicals that impact the endocrine system took a central stage as they have the potential to affect whole live cycle of humans through central functions like reproduction and metabolism, while accumulating over time both in the human body and the environment. Probably the best studied endocrine disrupting chemicals (EDCs) are the ones with estrogenic or anti-estrogenic activity. Of these, Bisphenol A (BPA) has garnered attention due to its widespread detection in the human population and the environment, observed effects in laboratory animals, and plausible impact on human health. BPA is among the top 2% of organic chemicals produced commercially and is used in a vast variety of applications including many day-to-day products containing polycarbonate plastics and epoxies. When BPA is used to make polymers for lining of cans or bottles, it often leaches out into food or beverages, and is then ingested in microgram quantities [1–3]. Given its prevalence, it is of little surprise that biomonitoring studies detected BPA in most humans [4]. Based on its mode-of-action and results from rodent studies, BPA has been implicated in hormone-dependent cancers (e.g., breast and prostate), metabolic diseases (e.g., diabetes and obesity), developmental defects, and changes in fertility, neurological function, and behavior [5–7]. In light of health and environmental concerns about the safety of BPA, the demand for non-BPA-containing materials is high and the search for viable BPA alternatives has grown dramatically. Unfortunately, bisphenol alternatives such as bisphenol S (BPS) and bisphenol F (BPF) have scant health and epidemiological data; however, they may have a similar mode-of-action to BPA, including endocrine disruption. In fact, a recent systematic review of 25 in vitro and 7 in vivo studies concluded that BPS and BPF have activity and potency that is similar to BPA with estrogen-, anti-estrogen-, androgen-, and anti-androgen-like features [8]. Finding BPA replacements that can be used in commercial applications such as coating material for canned food packaging is challenging since bisphenol polymers have inimitable physical properties that are not available from non-bisphenol materials such as olefins, acrylics or polyesters [9]. Bisphenol-based epoxy polymers are able to maintain integrity for long periods of time across a spectrum of applications, from easy open cans to twist-off bottle closures. Previously, no equivalent to bisphenol polymer chemistry had been found that is equal to BPA for preventing can lining failure and subsequent food contamination, especially with acidic or fatty foods [10]. Multiple cell-based in vitro assays have been developed to measure estrogenic potential of chemicals, with the most common being estrogen-dependent cell proliferation and gene expression. The measurement of estrogenic activity based upon breast cancer cell proliferation (E-screen assay) and luciferase-based reporter gene activation that is under the control of estrogen-responsive enhancer elements (BH1Luc4E2 cell line) has proven to be robust and highly sensitive, resulting in their consideration by multiple agencies (EPA, ICCVAM/NICEATM, and OECD) for national and international estrogenic activity standards [1,11]. However, individually, these assays provide little mechanistic insight to differentiate between similar chemical compounds with potential estrogenic activity. Recently, efforts from NIEHS and EPA has led to the development of mathematical models that incorporate data from 18 different ToxCast in vitro assays that measure ER binding, dimerization, DNA binding, transcriptional activation and cell growth [12]. Importantly, this model was shown to be able to accurately predict the results of the classical estrogenic in vivo mouse uterotrophic assay, thus leading towards a demonstration that in vitro high throughput (HT)/high content analysis (HCA) platforms could be used as a substitute for very expensive and slow animal models. Compounds such as BPA that mimic or antagonize the in vivo or in vitro activity of the hormone 17β-estradiol (E2) are typically described as having either estrogenic (agonistic) or anti-estrogenic (antagonistic) activity. This is largely mediated by interactions with one or both of the estrogen receptor subtypes, estrogen receptor-alpha (ERα) or estrogen receptor-beta (ERβ), which are ligand activated nuclear transcription factors. We previously described the generation of a human cell line containing a stable, microscopically-visible, multi-copy integration of the ER-responsive prolactin promoter-enhancer unit (PRL-HeLa; Supplemental Materials, S1 Fig). This cell line, following expression of GFP-ERα, allows for direct and simultaneous visualization and quantitation of ER DNA binding, recruitment of coregulators, epigenetic mark alterations, chromatin remodeling, and transcriptional regulation in response to ER ligands [13–15]. The PRL-HeLa cell line was further-adapted to high throughput microscopy-based screening by the generation of stable variants expressing either ERα or ERβ translationally-fused with green fluorescent protein (GFP-ERα:PRL-HeLa and GFP-ERβ:PRL-HeLa). When combined with our custom, automated image analysis platform [16], these cell lines were used to discriminate and classify the mechanistic effects of E2, ER antagonists, BPA and 18 closely related BPA analogs on ERα and ERβ, a dataset that has also been included in the EPA mathematical models [12,17,18]. In the present study, we use these ERα- and ERβ-expressing PRL-HeLa, the widely utilized ER-positive MCF-7 breast cancer, and the AR-positive LNCaP prostate cancer cell lines in high content/high throughput assays to characterize and classify seven, less well studied, potential BPA substitutes identified by Valspar during an internal yeast-based in vitro assay (data not published). The molecules that we analyzed include: 2,2’ BPA, bisguaiacol F (BGF), CHDM 4-hydroxybuyl acrylate (CHDM-4-HBA), hydroquinone (HydroQ), tetramethyl bisphenol F (TMBPF), tetramethyl bisphenol F diglycidyl ether (TMBPF-DGE), and TMBFP-ER (Fig 1). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Chemical structures and identification numbers for compounds analyzed. Each compound is identified by an abbreviation, full name, and a PubChem compound identification (or CAS) number. https://doi.org/10.1371/journal.pone.0180141.g001

Material and methods Chemicals Structures, abbreviations, and PubChem ID numbers for all compounds used in this study are shown in Fig 1. 17β-estradiol (E2, CAS 50-28-2), 4-hydroxytamoxifen (4HT, CAS 65213-48-1), bazedoxifene acetate (BZA, CAS 198481-33-3), and 2,2-bis(4-hydroxy-3,5-dimethylphenyl)propane (TM-BPA, CAS 227-033-5) were obtained from Sigma. Certified bisphenol A (BPA, CAS 80-05-7) was obtained from Battelle. 2,2’ BPA (CAS 7559-72-0) was obtained from Toronto Research Chemical. BGF (CASN 3888-22-0), CHDM-4-HBA (CAS N/A, PubChem CID 67072027), HydroQ (CAS 123-31-9), TMBPF (CAS 5384-21-4), TMBPF-DGE (CAS 93705-66-9), and TMBPF-ER (CAS 113693-69-9) were obtained from Valspar. These compounds are known reference compounds or investigational compounds previously identified as non-estrogenic using a yeast-based screening method performed internally by Valspar (unpublished data). All chemicals were solubilized in ethanol (E2, BPA, TM-BPA, 2,2’ BPA, BGF, TMBPF, TMBPF-ER, TMBPF-DGE) or DMSO (BZA, CHDM-4-HBA), aliquoted, and stored at -20°C until use. Cell culture and treatments The GFP-ERα:PRL-HeLa and GFP-ERβ:PRL-HeLa cell lines were grown in phenol red-free Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS; Gemini Bioproducts), L-glutamine, sodium pyruvate, 0.8 μg/ml blasticidin, 200 μg/ml hygromycin, and 10 nM 4HT (GFP-ERα:PRL-HeLa, Sigma) or raloxifene (GFP-ERβ:PRL-HeLa, Sigma). MCF-7 ERE-MAR cells were grown in phenol red-free DMEM containing 10% FBS, L-glutamine, and sodium pyruvate. 2PB-mCherry-NLS:LnCaP cells were grown in DMEM/F12 containing 10% FBS, L-glutamine, sodium pyruvate, 700 ng/ml puromycin, and 1 nM casodex. All cells were grown in treatment media (phenol red-free DMEM or DMEM/F12 containing 5% stripped/dialyzed FBS, L-glutamine, and sodium pyruvate and no drugs) for at least 48 hr prior to use. For PRL-Hela or 2PB-mCherry-NLS:LnCaP experiments, cells were robotically seeded using a Titertek dispenser in 384-well optical Cyclo-Olefin-Polymer (COP) bottom plates (384-IQ, Aurora Biotechnologies) that have been empirically-shown to be free of estrogenic activity. Cells were allowed to adhere overnight prior to treatment with compounds alone or in combination with 10 nM E2 or DHT at concentrations ranging from 50 pM to 5 μM for either 1 hr or 24 hr. For ERE-luciferase experiments, MCF-7 ERE-MAR cells (containing a stably integrated ERE-driven luciferase reporter gene) were manually plated into 24 well plates. Cells were allowed to adhere overnight prior to treatment with compounds alone at 2 μM and 5 μM. For combination experiments, cells were treated in the presence of 10 nM E2 for 24 hrs. Immunofluorescence Immunofluorescence was performed as previously described [17,19]. Cells were fixed in 4% EM-grade formaldehyde in PEM buffer (80 mM potassium PIPES [pH 6.8], 5 mM EGTA, and 2 mM MgCl 2 ) and quenched with 0.1 M ammonium chloride for 10 min. For samples in which no antibodies are used, cell membranes were permeabilized using 0.5% Triton X-100 for 10 min and DNA stained using DAPI (1 μg/ml) for 10 min. For samples with antibody labeling, permeabilization was with 0.5% Triton X-100 for 30 min. Cells were incubated at room temperature in blotto (5% milk in Tris-buffered saline/Tween 20) for 30 min, and then specific primary antibodies were added overnight at 4°C prior to 1 hr of secondary antibody (Alexa 647 conjugates; Molecular Probes) and DAPI staining (1 μg/ml for 10 min). The primary antibodies used were: mouse anti-Ser5-phospho RNA polymerase II (Abcam, ab5401) and mouse anti-SRC-3 (BD Transduction Labs No. 611105). High throughput microscopy and image analysis Image data sets were collected using the IC-200 image cytometer (Vala Sciences, San Diego, CA) utilizing dual-step high-speed (50–100 ms) reflection- and image-based autofocusing, either a 40X/0.95 Nikon S-fluor or a 20X/0.75 Plan Apo objective, and a sCMOS 5.5-megapixel camera and LED illumination. Z-stacks were collected at 1 μm (20X) or 0.5 μm (40x) intervals and maximum-projected using the instrument-supplied Mexican Hat 13 x 13 synthetic focus algorithm. Cell, nucleus, array segmentation and signal quantification was performed using the myImageAnalysis web application powered by Pipeline Pilot software (Biovia) as previously described [16]. Aggregated cells, mitotic cells, and apoptotic cells were removed using filters based on nuclear size, nuclear shape, and nuclear intensity. MCF-7 ERE-MAR luciferase assay MCF-7 ERE-MAR (also known as MCF-7-XVA2-Luc) cells, a kind gift from Dr. Steffi Oesterreich (University of Pittsburgh), are a human breast cancer cell line that contains a stable integration of the firefly luciferase gene under control of a promoter containing the estrogen response element (ERE) and matrix attachment region (MAR) derived from the Xenopus vitellogenin A2 promoter and responds to estrogenic compounds with a 30-35-fold induction of firefly luciferase [20]. After 24 hr of compound treatment, culture medium was aspirated and plates were frozen at -20°C until further processing. Cells were lysed with 1% Triton X-100, 10% glycerol, 2 mM EGTA, and 1 mM DTT. Luciferase was then measured using an automated microplate luminometer with the Promega Luciferase Assay System. Results from 4 replicates were collected and normalized to controls wells treated with E2 only. Calculation of estrogenicity (%E2) and statistical analysis The estrogenicity of compounds was calculated relative to the maximum response observed with 10 nM E2 treatment (%E2, a measure of response amplitude). Each compound at each concentration was measured in triplicate or quadruplicate. Basal activity of each assay was determined using “mock” control (MC) containing treatment media and vehicle controls (VC) containing 0.5% DMSO solvent in a minimum of eight samples on each assay plate. The MC was set to 0% resulting in a %E2 calculation (1): (1) Typical values for VC were 0% ± 5% E2. Inclusion of a VC accounts for any extraneous residual estrogenicity that might exist in the media or derived from the materials used for sample preparation. A chemical is considered to be significantly different from VC controls if it produces a mean %E2 significantly greater (p < 0.05) than VC as determined by ANOVA and post-hoc pairwise comparisons made using the Tukey HSD method. A compound is considered to have inhibited the E2 induced response if, when added to 10nM E2, it produces a mean %E2 significantly less than E2 controls. As a general rule, we consider a compound to have significant estrogenic properties with a %E2 score greater than 10% when used alone or anti-estrogenic properties with a %E2 change greater than 15%. %DHT calculations were done in an identical manner using 10 nM DHT at the control response. Averaged results from a minimum of 4 independent experiments were graphed ± standard deviation (SD) using SigmaPlot software with EC 50 values determined by fitting a 4-parameter sigmoidal curve. All data used to generate summary data can be found in a supplemental data file (S1 Data). Clustering analysis was performed using Cluster 3.0 and visualized with Java Treeview.

Discussion In response to concerns regarding BPA, there is an emphasis on manufacturing products that no longer use BPA. In some cases, BPA has been replaced with analogs such as BPS and BPF; consequently, human exposure to these analogs is now approaching the same levels as BPA [8]. It is becoming clear that BPA analogs can have similar effects as BPA in cell-based in vitro assays and in vivo animal models [8,17,24,26,27]. These results emphasize the need for rigorous studies to understand potential endocrine effects of compounds proposed as BPA alternatives before widespread adoption. To this end, we completed a set of cell-based studies that included quantitative and mechanistic metrics of estrogenic and androgenic activity on a set of compounds previously identified using a yeast-based in vitro screening approach as having minimal estrogen-like properties. When utilizing the PRL-HeLa and MCF-7 cell models to characterize a set of reference compounds and seven investigational compounds (2’,2 BPA, BGF, CHDM-4-HBA, HydroQ, TMBPF, TMBPF-DGE, and TMBPF-ER), the estrogenic effects of BPA were clear and consistent with previous work. In PRL-HeLa cells expressing either GFP-ERα or GFP-ERβ, BPA exposure resulted in an extent of visible array formation comparable with E2 treatment. This indicates that at a sufficient concentration, BPA exposure results in the efficient binding of ER (α or β) to the estrogen response sequences located in the prolactin promoter, a crucial step in estrogen regulated gene expression. This result was confirmed by the significant induction of de novo mRNA production at the PRL array and ERE-driven reporter gene activity in the estrogen responsive MCF-7 breast cancer cell line. In contrast, none of the seven investigational compounds were able to induce significant promoter binding in PRL-HeLa cells expressing either GFP-ERα or GFP-ERβ when tested up to micromolar concentrations at both short (1 hr) and long (24 hr) time points. Based upon hierarchical clustering using the assays presented, these 7 compounds populate a cluster distinct from BPA and 18 other previously- studied BPX compounds and are marked by a relative lack of estrogenic activity (Fig 5). Within in this cluster, 2,2’ BPA is unique in that it is characterized by weak estrogenic activity in the MCF-7 cell line with little to no activity in the PRL-HeLa cell lines. This indicates that the altered position of the hydroxyl groups results in a receptor-ligand interaction that retains residual activity on the basic ERE element contained in the MCF-7 reporter cell line, but is insufficient to drive estrogenic activity in the context of the prolactin promoter elements used in the PRL-HeLa cell lines. Unique within this cluster are the responses observed with CHDM-4-HBA and TMBPF. These are the first compounds we have observed that demonstrated any ability to alter the promoter binding induced by 10 nM E2, reducing ERα recruitment to the PRL array by ~80% and ~40% and significantly reducing de novo mRNA production. This is in contrast to well-studied compounds with anti-estrogenic activity such as 4HT and BZA, which antagonize the E2 transcriptional response by the recruitment of corepressor complexes that inhibit transcription, but do not alter ERα recruitment to the PRL array [15,17]. The effect was greatest in the GFP-ERα:PRL-HeLa cell line and not observed or significantly reduced with the GFP-ERβ:PRL-HeLa and the MCF-7 cell lines. In addition, only TMBPF demonstrated an effect on the androgen induced transcriptional activity of the promiscuous AR receptor expressed in the LNCaP prostate cancer cell line. These results suggest that these effects on transcriptional activity are selective based upon receptor, receptor subtype preference, and/or cell background. When promoter binding was observed, neither compound altered the recruitment of the ER coregulator SRC-3 and only CHDM-4-HBA decreased levels of activated RNA Pol II at the integrated PRL array. One potential mechanism by which these compounds may be acting is through the activation of other type I nuclear receptors, as others have observed that activation of the glucocorticoid receptor GR can displace ER from DNA binding sites [28]. The PRL-HeLa cell line does express endogenous GR; however, we have shown that direct activation of endogenous GR using dexamethasone increases, not decreases, ERα recruitment to the PRL array which suggests this mechanism is not involved in the responses observed with CHDM-4-HBA and TMBPF [19]. Therefore, the mechanisms behind these observations remain unknown, and importantly, it has been subsequently shown that TMBPF and its related compounds do not show biological effects in either E-SCREEN or mouse uterotorphic in vivo assays [29]. The remaining four investigational compounds, including two TMBPF derivatives (BGF, TMBPF-DGE, TMBPF-ER, and HydroQ), demonstrated little activity in the in vitro assays for estrogenic, anti-estrogenic, androgenic, and anti-androgenic activity. Although a significant decrease in E2 induced reporter gene activity in the MCF-7 cell line was observed with 5 μM TMBPF-DGE, TMBPF-ER, and HydroQ, significant cell toxicity was also observed. When the concentration was reduced from 5 μM to 2 μM, cell toxicity was absent, and there was no observable effect on E2 induced reporter gene activity. This suggests that the results observed at the higher tested concentration were due to non-specific cell toxicity and not a specific anti-estrogenic-like activity associated with the compounds. Although CHDM-4-HBA appeared to demonstrate anti-estrogenic-like activity in the PRL-HeLa assays, this result was not supported in the ERE-luciferase assays performed using the MCF-7 cell line that endogenously expresses ERα. Considering these findings, it appears several of these investigational compounds, especially HydroQ, BGF and the TMBPF derivatives TMBPF-DGE and TMBPF-ER, demonstrate little of the estrogen-like activity shown by BPA and various BPA analogs. The TMBPF findings are consistent with a recent publication in which this compound was found to not have estrogenic-like properties in an in vivo uterotrophic assay [29]. HydroQ, a compound with evidence of weak anti-estrogen-like activity in the GFP-ERβ:PRL-HeLa cell line, is the only investigational compound that has been included in the EPA ToxCast program where it was found to have weak estrogenic activity at concentrations (EC 50 > 30 μM) exceeding those tested in this study [30]. There is little to no existing data describing the estrogenic or anti-estrogenic activity of BGF, TMBFP-DGE and TMBPF-ER, compounds that may represent useful alternatives to BPA. For example, the lack of activity by BGF is exciting as this is a “green” compound readily synthesized from plant material found in by-products from the paper industry [31]. Based upon the lack of activities demonstrated here and minimal pre-existing data, these compounds are worthwhile candidates for further investigation using an expanded panel of in vitro assays such as those sanctioned by the EPA and used in current models predicting estrogenic activity [12], and/or using in vivo models to further our understanding as suitable BPA alternatives.

Supporting information S1 Data. Source and raw data for summary figures. Excel worksheet containing oringal and normalized data used to generate summary figures. https://doi.org/10.1371/journal.pone.0180141.s001 (XLSX) S1 Fig. Prolactin integrated promoter array model system. (A) Schema showing the essential elements of the reporter constructions. Transcription start site, proximal promoter and enhancer sequence are shown. (B) Multi-copy integration in a HeLa cell line stably expressing GFP-tagged ER allows visualization of estrogen induced binding as a bright intra-nuclear spot of varying size/shape/texture linked to transcriptional activity (Bolt et al, 2014; Stossi et al, 2014). Red lines indicate array mask (1) and cell mask (2) generated by image analysis algorithms and allow quantification of features listed. Examples of samples treated with either 10 nM estradiol (C) or non-estrogenic 5 μM 2,2’ BPA (D). https://doi.org/10.1371/journal.pone.0180141.s002 (TIF)

Acknowledgments This work was supported by NIEHS grants NIEHS R01 (1R01ES023206-01; Cheryl L. Walker, Bert W. O’Malley, M.A.M. and Mark T. Bedford) and NIEHS P30 (ES023512-01; Center of Excellence in Environmental Health, C.L.W.). Further support was provided by the Integrated Microscopy Core at Baylor College of Medicine with funding from the John S. Dunn Gulf Coast Consortium for Chemical Genomics, the Dan L. Duncan Cancer Center (NIH P30CA125123), and the NIH (HD007495, and DK56338). ATS is a K12 Scholar supported by NIH grant K12DK0083014, the multidisciplinary K12 Urologic Research (KURe) Career Development Program awarded to Dr. Dolores J. Lamb. DeepBio, Inc received funds and material support from the Valspar Corporation. Funders do not have scientific or editorial control over the design, conduct, analysis, or interpretation of the data, nor were they involved in the decision to publish or producing this manuscript. DeepBio, Inc provided support in the form of materials and consulting fees for A.T.S., F.S., and M.G.M. and provided salary for M.A.M. The specific roles of these authors are articulated in the ‘author contributions’ section. M.A.M participated in in study design, data interpretation, and final manuscript review, however, DeepBio, Inc did not have editorial control nor was involved in the decision to publish.

Author Contributions Conceptualization: ATS FS CLW MAM. Data curation: ATS FS. Formal analysis: ATS. Funding acquisition: CLW MAM. Investigation: ATS FS MGM. Methodology: ATS FS MGM. Project administration: ATS MGM MAM. Resources: CLW MAM. Software: ATS. Supervision: CLW MAM. Validation: ATS FS MGM. Visualization: ATS. Writing – original draft: ATS. Writing – review & editing: ATS FS CLW MAM.