Assay Readout Research Articles

Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement

Determination of influenza-specific antibody titers is commonly done using the hemagglutination inhibition assay (HAI) and the viral microneutralization assay (MN). Both assays are characterized by high intra- and inter-laboratory variability. The HAI assay offers little opportunity for standardization. For the MN assay, variability might be due to the use of different assay protocols employing different readouts. We therefore aimed at investigating which of the MN assay readout methods currently in use would be the most suitable choice for a standardized MN assay that could serve as a substitute for the HAI assay. For this purpose, human serum samples were tested for the presence of influenza specific neutralizing antibodies against A/California/7/09 H1N1 (49 sera) or A/Hong Kong/4801/2014 (50 sera) using four different infection readout methods for the MN assay (cytopathic effect, hemagglutination, ELISA, RT qPCR) and using the HAI assay. The results were compared by correlation analysis and by determining the level of agreement before and after normalization to a standard serum. Titers as measured by the 4 MN assay readouts showed good correlation, with high Person’s r for most comparisons. However, agreement between nominal titers varied with readouts compared and virus strain used. In addition, Pearson’s correlation of MN titers with HAI titers was high but agreement of nominal titers was moderate and the average difference between the readings of two assays (bias) was virus strain-dependent. Normalization to a standard serum did not result in better agreement of assay results. Our study demonstrates that different MN readouts result in nominally different antibody titers. Accordingly, the use of a common and standardized MN assay protocol will be crucial to minimize inter-laboratory variability. Based on reproducibility, cost effectiveness and unbiased assessment of results we elected the MN assay with ELISA readout as most suitable for a possible replacement of the HAI assay.

Aggregation of chitosan nanoparticles in cell culture: Reasons and resolutions.

Nanoparticles are promising drug delivery systems which are flexible for targeting specific tissues to reduce therapeutic doses and minimize side effects. Nanoparticles should be maintained with high stability and uniformity; however, aggregation is a major challenge which commonly impairs stability and efficacy of nanocarriers. In this study, we revisited the factors that influence the stability of chitosan (Protasan™ UP CL113) nanoparticles prepared with ionotropic gelation, widely recognized to be prone to aggregation, and proposed a model to overcome the negative influence of aggregation while testing in vitro efficacy. Decrease in pH due to cell proliferation, 37°C cell culture temperature, serum in culture media, and incubation time were considered as factors causing chitosan nanoparticles' aggregation which deteriorates cell culture assay readouts, increases optical density values and leads to false-positive results. Size and stability studies were not sufficient to avoid misleading results in cell culture. The chitosan nanoparticle aggregation was almost inevitable under standard culture conditions; nevertheless, the removal of nanoparticles before aggregation but after an incubation period long enough for efficient cellular uptake was determined as a feasible and inexpensive method for testing the in vitro efficacy of polymeric nanoformulations. This approach was used with blank and gemcitabine-loaded chitosan nanoparticles on pancreatic cancer cells and proved to be useful for reliable cytotoxicity results.

A high throughput zebrafish chemical screen reveals ALK5 and non-canonical androgen signalling as modulators of the pkd2\u2212/\u2212 phenotype

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of end-stage renal failure in humans and results from germline mutations in PKD1 or PKD2. Despite the recent approval of tolvaptan, safer and more effective alternative drugs are clearly needed to slow disease progression. As a first step in drug discovery, we conducted an unbiased chemical screen on zebrafish pkd2 mutant embryos using two publicly available compound libraries (Spectrum, PKIS) totalling 2,367 compounds to identify novel treatments for ADPKD. Using dorsal tail curvature as the assay readout, three major chemical classes (steroids, coumarins, flavonoids) were identified from the Spectrum library as the most promising candidates to be tested on human PKD1 cystic cells. Amongst these were an androgen, 5α−androstane 3,17-dione, detected as the strongest enhancer of the pkd2 phenotype but whose effect was found to be independent of the canonical androgen receptor pathway. From the PKIS library, we identified several ALK5 kinase inhibitors as strong suppressors of the pkd2 tail phenotype and in vitro cyst expansion. In summary, our results identify ALK5 and non-canonical androgen receptors as potential therapeutic targets for further evaluation in drug development for ADPKD.

In-depth comparison of cell-based methodological approaches to determine drug susceptibility of visceral Leishmania isolates.

Monitoring the drug susceptibility of Leishmania isolates still largely relies on standard in vitro cell-based susceptibility assays using (patient-isolated) promastigotes for infection. Although this assay is widely used, no fully standardized/harmonized protocol is yet available hence resulting in the application of a wide variety of host cells (primary cells and cell lines), different drug exposure times, detection methods and endpoint criteria. Advocacy for standardization to decrease inter-laboratory variation and improve interpretation of results has already repeatedly been made, unfortunately still with unsatisfactory progress. As a logical next step, it would be useful to reach at least some agreement on the type of host cell and basic experimental design for routine amastigote susceptibility determination. The present laboratory study using different L. infantum strains as a model for visceral leishmaniasis species compared primary cells (mouse peritoneal exudate (PEC), mouse bone marrow derived macrophages and human peripheral blood monocyte derived macrophages) and commercially available cell lines (THP-1, J774, RAW) for either their susceptibility to infection, their role in supporting intracellular amastigote multiplication and overall feasibility/accessibility of experimental assay protocol. The major findings were that primary cells are better than cell lines in supporting infection and intracellular parasite multiplication, with PECs to be preferred for technical reasons. Cell lines require drug exposure of >96h with THP-1 to be preferred but subject to a variable response to PMA stimulation. The fast dividing J774 and RAW cells out-compete parasite-infected cells precluding proper assay read-out. Some findings could possibly also be applicable to cutaneous Leishmania strains, but this still needs cross-checking. Besides inherent limitations in a clinical setting, susceptibility testing of clinical isolates may remain problematic because of the reliance on patient-derived promastigotes which may exhibit variable degrees of metacyclogenesis and infectivity.

Abstract C116: Phenotypic strategies for anticancer drug discovery

Abstract Although recent innovations in anti-cancer therapies have led to better treatment strategies and improved patient outcomes, cancer is still one of the leading causes of morbidity and mortality, globally. Thus, the need to pursue better and safer therapies remains as critical and compelling as ever. Improving and streamlining drug development efforts in this area will expedite the progress already made in offering new treatment modalities for cancer patients. By incorporating more translational, predictive approaches earlier in the drug discovery pipeline, we can enable the identification of more efficacious and safer pre-clinical candidates to advance to the clinic. Eurofins Discovery BioMAP® (CRC) Oncology Panel models human tumor-immune microenvironment (TME) biology in vitro by co-culturing host stromal or vascular cell types with PBMC and the HT-29 human tumor cell line. Standard assay readouts include 42 protein biomarkers that inform on a test agent’s impact on the complex TME co-culture. We sought to expand these models to include a measure of cancer cell-specific killing relevant for clinical efficacy. Briefly, we screened a select number of reference compounds on a set of ≥240 cancer cell lines (including HT-29) available in the OncoPanel™ assay in order to select agents and concentrations specific for direct HT-29 killing. The HT-29 cells were then pre-loaded with a cell membrane-penetrant fluorescent dye, and incorporation was verified by flow cytometry. Labelled HT-29 cells were then added to a fibroblast/PBMC or HuVEC/PBMC co-culture, and the assay was performed according to our standard protocol. Briefly, test agent addition was followed by stimulation to recapitulate immune activation relevant for the TME. After 48 hours, protein-based biomarkers were measured via ELISA or electrochemiluminescence, and cancer cell death was visualized by high-content imaging. Imaging of the fluorescently-labeled HT-29 cells showed zero transfer of the dye to the host cell types present in the systems. Additionally, the TME biomarker profiles were unaffected by the incorporation of the dye into the HT-29 cells. Profiling test agents in the OncoPanel assay allows for rapid stratification of cancer cell types for sensitivity. Subsequent profiling in BioMAP Oncology systems allows for assessment of test agent impacts on the physiologically relevant TME. Here we demonstrate the ability to detect cancer cell viability within the context of the TME, either by direct action of the anti-cancer agent or indirectly via the immune-restorative effects of a test agent relevant for immune-oncology therapies. These translational assays can enable the fast and effective prioritization of test agents based on concurrent assessment of efficacy and safety towards better clinical candidates and improved patient outcomes. Citation Format: Steven M. Garner, Alyssa M. Croff, Justin H. Lipner, Alison O'Mahony, Sharlene Velichko, Alastair J. King, Jesse J. Parry. Phenotypic strategies for anticancer drug discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C116. doi:10.1158/1535-7163.TARG-19-C116

Paper Stacks for Uniform Rehydration of Dried Reagents in Paper Microfluidic Devices

Spatially uniform reconstitution of dried reagents is critical to the function of paper microfluidic devices. Advancing fluid fronts in paper microfluidic devices drive (convect) and concentrate rehydrated reagents to the edges, causing steep chemical gradients and imperfect mixing. This largely unsolved problem in paper microfluidics is exacerbated by increasing device dimensions. In this article, we demonstrate that mixing of dried reagents with a rehydrating fluid in paper microfluidics may be significantly enhanced by stacking paper layers having different wicking rates. Compared to single-layer paper membranes, stacking reduced the “non-reactive area”, i.e. area in which the reconstituted reagents did not interact with the rehydrating fluid, by as much as 97% in large (8 cm × 2 cm) paper membranes. A paper stack was designed to collect ~0.9 ml liquid sample and uniformly mix it with dried reagents. Applications of this technology are demonstrated in two areas: (i) collection and dry storage of sputum samples for tuberculosis testing, and (ii) salivary glucose detection using an enzymatic assay and colorimetric readout. Maximizing the interaction of liquids with dried reagents is central to enhancing the performance of all paper microfluidic devices; this technique is therefore likely to find important applications in paper microfluidics.

Label-Free Estimation of Therapeutic Efficacy on 3D Cancer Spheres Using Convolutional Neural Network Image Analysis.

Despite recent advances in cancer treatment, developing better therapeutic reagents remains an essential task for oncologists. To accurately characterize drug efficacy, 3D cell culture holds great promise as opposed to conventional 2D monolayer culture. Due to the advantages of cell manipulation in high-throughput, various microfluidic platforms have been developed for drug screening with 3D models. However, the dissemination of microfluidic technology is overall slow, and one missing part is fast and low-cost assay readout. In this work, we developed a microfluidic chip forming 1920 tumor spheres for drug testing, and the platform is supported by automatic image collection and cropping for analysis. Using conventional LIVE/DEAD staining as the ground truth of sphere viability, we trained a convolutional neural network to estimate sphere viability based on its bright-field image. The estimated sphere viability was highly correlated with the ground truth (R-value > 0.84). In this manner, we precisely estimated drug efficacy of three chemotherapy drugs, doxorubicin, oxaliplatin, and irinotecan. We also cross-validated the trained networks of doxorubicin and oxaliplatin and found common bright-field morphological features indicating sphere viability. The discovery suggests the potential to train a generic network using some representative drugs and apply it to many different drugs in large-scale screening. The bright-field estimation of sphere viability saves LIVE/DEAD staining reagent cost and fluorescence imaging time. More importantly, the presented method allows viability estimation in a label-free and nondestructive manner. In short, with image processing and machine learning, the presented method provides a fast, low-cost, and label-free method to assess tumor sphere viability for large-scale drug screening in microfluidics.

Dissecting phenotypic responses of the druggable targetome in cancers

Although a large amount of screening data comprising target genes and/or drugs tested against cancer cell line panels are available, different assay conditions and readouts limit the integrated analysis and batch-to-batch comparison of these data. Here, we systematically produced and analyzed the anticancer effect of the druggable targetome to understand the varied phenotypic outcomes of diverse functional classes of target genes. A library of siRNAs targeting ~4,800 druggable genes was screened against cancer cell lines under 2D and/or 3D assay conditions. The anticancer effect was simultaneously measured by quantifying cell proliferation and/or viability. Hit rates varied significantly depending on assay conditions and/or phenotypic readouts. Functional classes of hit genes were correlated with the microenvironment difference between the 2D monolayer cell proliferation and 3D sphere formation assays. Furthermore, multiplexing of cell proliferation and viability measures enabled us to compare the sensitivity and resistance responses to the gene knockdown. Many target genes that inhibited cell proliferation increased the single-cell-level viability of surviving cells, leading to an increase in self-renewal potential. In this study, combinations of parallel 2D/3D assays and multiplexing of cell proliferation and viability measures provided functional insights into the varied phenotypic outcomes of the cancer targetome.

High-Content Screening for Cryptosporidium Drug Discovery.

High-content screening (HCS) is a cell-based type of phenotypic screening that combines multiple simultaneous readouts with a high level of throughput. A particular benefit of this form of screening for drug discovery is the ability to perform the interrogation in a biologically relevant system. This approach has greatly advanced the field of drug discovery for cryptosporidiosis, a diarrheal disease caused by protozoan parasites of Cryptosporidium spp. These parasites are obligate intracellular parasites and cannot be cultured in vitro without the support of a host cell, limiting the options for potential assay readout. Here we describe an established 384- or 1536-well format high-content imaging (HCI) assay of Cryptosporidium-infected HCT-8 human ileocecal adenocarcinoma cells. This HCS assay is a powerful tool to assess large numbers of compounds to power drug discovery, as well as to phenotypically characterize known Cryptosporidium-active compounds.

Engineering genetically encoded FRET-based nanosensors for real time display of arsenic (As3+) dynamics in living cells

Arsenic poisoning has been a major concern that causes severe toxicological damages. Therefore, intricate and inclusive understanding of arsenic flux rates is required to ascertain the cellular concentration and establish the carcinogenetic mechanism of this toxicant at real time. The lack of sufficiently sensitive sensing systems has hampered research in this area. In this study, we constructed a fluorescent resonance energy transfer (FRET)-based nanosensor, named SenALiB (Sensor for Arsenic Linked Blackfoot disease) which contains a metalloregulatory arsenic-binding protein (ArsR) as the As3+ sensing element inserted between the FRET pair enhanced cyan fluorescent protein (ECFP) and Venus. SenALiB takes advantage of the ratiometic FRET readout which measures arsenic with high specificity and selectivity. SenALiB offers rapid detection response, is stable to pH changes and provides highly accurate, real-time optical readout in cell-based assays. SenALiB-676n with a binding constant (Kd) of 0.676 × 10−6 M is the most efficient affinity mutant and can be a versatile tool for dynamic measurement of arsenic concentration in both prokaryotes and eukaryotes in vivo in a non-invasive manner.

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.

Further exploration of an N-aryl phenoxyethoxy pyridinone-based series of mGlu3 NAMs: Challenging SAR, enantiospecific activity and in vivo efficacy.

This letter describes the further optimization of a series of mGlu3 NAMs based on an N-aryl phenoxyethoxy pyridinone core. A multidimensional optimization campaign, with focused matrix libraries, quickly established challenging SAR, enantiospecific activity, differences in assay read-outs (Ca2+ flux via a promiscuous G protein (Gα15) versus native coupling to GIRK channels), identified both full and partial mGlu3 NAMs and a new in vivo tool compound, VU6017587. This mGlu3 NAM showed efficacy in tail suspension, elevated zero maze and marble burying, suggesting selective inhibition of mGlu3 affords anxiolytic-like and antidepressant-like phenotypes in mice.

A Robust and Scalable High-Throughput Compatible Assay for Screening Amyloid-β-Binding Compounds.

A robust fluorescent readout assay using topologically-sensitive dyes improves the screening of novel amyloid-binding molecules. One of the key components that make this assay more realistic is the use of endogenous amyloid obtained from 5XFAD mouse brains. The assay conditions were optimized for high throughput screening operation with Z-prime values >0.6. Using a combination of library of 3,500 compounds including known drugs, natural-derived molecules and random organic molecules, 8 unique molecules were identified as potential amyloid-binding agents.

Abstract 34: A high-content imaging platform for quantitative assessment of immunogenic tumor cell killing and T-cell proliferation in 3D tumoroids of fresh patient tumor samples

Abstract Introduction: Challenges in the development of immunotherapeutics still remain due to the lack of clinically relevant drug screening platforms that recapitulate the complexity of the tumor immune microenvironment. T-cell activation and immunogenic tumor cell death have been the most desired assay readouts to fully explore the efficacy of immuo-oncology drugs and to develop most effective rational drug combinations. Conventional cytotoxicity assays such as Cr51 and LDH release assays are limited in providing clinically relevant data about treatment mediated tumor cell killing by immune cells. Here, we describe a novel 3D live tumor explant model of fresh patient tumor tissue (3D-EXplore) to simultaneously and cost effectively evaluate T-cell activation and tumor cell killing by immuno-oncology drugs. Materials and Methods: All human tumor samples were obtained with patients’ consent and relevant IRB approval. Fresh patient tumor samples of various cancer types (lung, bladder, kidney) were processed to generate unpropagated 3D tumoroids of standard size (100-150 microns). 3D tumoroids were treated with pembrolizumab (Keytruda) and atezolizumab (Tecentriq) alone or in combination with conventional chemotherapeutic drugs. Various combinations of viability, vitality, cell proliferation and cell death fluorogenic dyes were utilized to label tumor cells prior to high content confocal imaging. Treatment-mediated changes in tumor cell death and T-cell activation were quantified by Nilogen’s QTDI-algorithm. The results were corroborated by multiplex flow cytometry and cytokine relase assays. Results: 3D-tumoroids were successfully prepared from different tumor types. Tumor cell viability and immune cell composition including CD4 and CD8 T-cells, Tregs, NK cells and M1/M2 macrophages were monitored over 5 days to ensure obtaining reliable results. Using high-content image analysis and flow cytometric analysis, in selected tumors we observed a significant increase in T-cell activation and tumor cell killing upon ex vivo treatment with pembrolizumab or atezolizumab alone, which was enhanced in the presence of carboplatin. Conclusion: Here we have described a cost-effective and rapid drug testing platform using clinically relevant unpropogated fresh patient tumor tissue for rapid testing of multiple drugs and drug combinations based on tumor cell killing and T-cell activation to accelerate drug discovery, which can be applied in clinical studies to improve personalized immunotherapy for individual patients. Citation Format: Melanie Mediavilla-Varela, Vijayendra Agrawal, Melba Marie Page, Jenny Kreahling, Soner Altiok. A high-content imaging platform for quantitative assessment of immunogenic tumor cell killing and T-cell proliferation in 3D tumoroids of fresh patient tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 34.

Abstract 2187: Tryptophan Metabolism Plays a Central Role in Immunosuppression

Abstract Tryptophan metabolism plays a central role in immunosuppression through the local depletion of tryptophan with the concomitant production and accumulation of kynurenine, both of which are immunosuppressive. Indoleamine 2,3-dioxygenase 1 (IDO1), a heme-dependent enzyme, catalyzes the initial and rate-limiting step of the kynurenine pathway. Tumor cells selectively upregulate IDO1 as an immune-evasion mechanism either through intrinsic expression of IDO1, or in response to IFN-γ, a cytokine secreted by immune cells during an active immune response. In addition to the immuno-suppressive role of IDO1 and kynurenine, we investigated their additional roles in tumor cells. Here we describe an NMR-based readout assay for IDO1 activity, in which we are able to trace the extra- and intracellular destination of the single tryptophan carbon groups. This assay allowed us to detect contribution of the tryptophan catabolism to purine synthesis in tumor cells, and suggested further roles of tryptophan catabolism in tumors, apart from kynurenine production, that contribute to the modulation of tumor growth. We detected the incorporation of labeled tryptophan carbon units in intracellular purine pools. Our highly potent, orally available, and CNS penetrant small molecule inhibitor of IDO1 (LY3381916) inhibits this incorporation of tryptophan-derived one carbon units into purines in vitro and in vivo. This activity leads to pre-clinical efficacy and may be an added advantage of inhibiting IDO1 in cancer. Citation Format: Sandaruwan Geeganage, Lillian Sams, James Henry, Frank Dorsey, Kenneth Roth, Alexander Nikolayev, Karim Benhadji, Raymond Gilmour, Ana Cerezo, Sandra Peregrina, Gloria Martínez-del Hoyo, Ramón Campos-Olivas, Juan Manuel Funes, Laura Diezma, Susana Velasco-Miguel. Tryptophan Metabolism Plays a Central Role in Immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2187.

Abstract 2014: A novel small molecule inhibitor to suppress Notch1 activation in CRC

Abstract Introduction: Earlier we reported that aberrant overexpression of Notch1 in colon cancer patients is responsible for aggressiveness and progression of the disease. Current, therapeutic interventions of notch1 inhibitors that binds catalytic unit of γ-secretase and disrupts the cleavage activity cause gastrointestinal toxicity in clinical trial. Therefore, our study aims to develop a small molecule inhibitor, which inhibits Notch1 signaling but binds outside of γ-secretase’s catalytic unit thereby substantially reducing associated toxicity attributes. Through structure-activity relationship (SAR) studies based on naturally occurring Withaferin A (WA) structure, we have recently identified a WA-difuroate analog ASR458 as one such small molecule. Methods: To analyze the effect of ASR490 on HCT116 and HCT116/Notch1 cells, we performed phenotypic, western blot and in vivo analysis. Results: Notch1 overexpressing CRC cells (HCT/Notch1) showed aggressive cell growth (42%) as compared to HCT116 cells, however, ASR490 treatment inhibited cell growth in both HCT116 (IC50: 600 nM) as well as HCT/Notch1 cells (IC50: 850 nM). ASR458 significantly inhibited Notch1 expression and its downstream events in both the transfected cell lines. As a result, we observed shutdown of pro-survival machinery and induction of pro-apoptotic events (Bax, caspases and PARP) resulting in apoptotic cell death in ASR456 treated HCT116 and Notch1/HCT116 cells. Since Notch1 is responsible for epithelial mesenchyme transition (EMT); we analyzed EMT drivers and readout invasion/migration assays. ASR458 treatment reversed the mesenchymal characteristics by restoring the function of E-cadherin, which resulted in the suppression of invasion and migration in readout assays of HCT116 transfectants. Our ongoing Xenotransplanted mice experiments may suggest whether ASR458 inhibits the growth of HCT116 and HCT116/Notch1 tumors. Conclusion: Restrained gastrointestinal toxicity of Notch1 targeting therapeutics is achievable with novel small molecules displaying a differential binding affinity to γ-secretase. ASR458 inhibits cell growth in Notch1 overexpressing cells and abrogates Notch1 induced EMT. Ongoing molecular docking and in vivo studies may confirm ASR458 binding sites in γ-secretase and validate our in vitro findings respectively. Citation Format: Chendil Damodaran, Ashish Tyagi, Balaji Chandrasekaran, Venkatesh Kolluru, Ibrahim Jojua, Srinivasa Ramisetti, Arun K. Sharma, Murali K. Ankem. A novel small molecule inhibitor to suppress Notch1 activation in CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2014.

Leveraging heterogeneous data from GHS toxicity annotations, molecular and protein target descriptors and Tox21 assay readouts to predict and rationalise acute toxicity

Despite the increasing knowledge in both the chemical and biological domains the assimilation and exploration of heterogeneous datasets, encoding information about the chemical, bioactivity and phenotypic properties of compounds, remains a challenge due to requirement for overlap between chemicals assayed across the spaces. Here, we have constructed a novel dataset, larger than we have used in prior work, comprising 579 acute oral toxic compounds and 1427 non-toxic compounds derived from regulatory GHS information, along with their corresponding molecular and protein target descriptors and qHTS in vitro assay readouts from the Tox21 project. We found no clear association between the results of a FAFDrugs4 toxicophore screen and the acute oral toxicity classifications for our compound set; and a screen using a subset of the ToxAlerts toxicophores was also of limited utility, with only slight enrichment toward the toxic set (odds ratio of 1.48). We then investigated to what degree toxic and non-toxic compounds could be separated in each of the spaces, to compare their potential contribution to further analyses. Using an LDA projection, we found the largest degree of separation using chemical descriptors (Cohen’s d of 1.95) and the lowest degree of separation between toxicity classes using qHTS descriptors (Cohen’s d of 0.67). To compare the predictivity of the feature spaces for the toxicity endpoint, we next trained Random Forest (RF) acute oral toxicity classifiers on either molecular, protein target and qHTS descriptors. RFs trained on molecular and protein target descriptors were most predictive, with ROC AUC values of 0.80–0.92 and 0.70–0.85, respectively, across three test sets. RFs trained on both chemical and protein target descriptors combined exhibited similar predictive performance to the single-domain models (ROC AUC of 0.80–0.91). Model interpretability was improved by the inclusion of protein target descriptors, which allow the identification of specific targets (e.g. Retinal dehydrogenase) with literature links to toxic modes of action (e.g. oxidative stress). The dataset compiled in this study has been made available for future application.

Electrostatic Read Out for Label-Free Assays Based on Kelvin Force Principle

Detection methods and analytical devices have drawn increasing attention in recent years due to their direct impact on early detection, monitoring and diagnosis of disease in medical research. In this work, we describe a simple but so far unrecognized label-free method for surface-bound analyte detection, which could be applicable to a wide range of substances. In this respect, the feasibility and practical aspects of a micrometer-scale, poly-l-lysine-based, solid-phase assay for label-free analyte detection with electrostatic read-out are investigated. Micropatterned poly-l-lysine layers were produced using soft-lithography on mica and their electrostatic surface potential was determined using Kelvin-probe Force Microscopy. Ribose, a natural sugar, was used as analyte. Upon exposure to ribose, the surface potential changed from positive to negative in a reversible manner. We report for the first time the use of an electrostatic principle for assay read-out. This purely physical effect could be used to develop label- and marker-free assays for sugars, various other substances or, possibly, biosensors.Graphical

Continuous Readout versus Titer-Based Assays of Influenza Vaccine Trials: Sensitivity, Specificity, and False Discovery Rates.

The current gold standard for measuring antibody-based immunity to influenza viruses relies on the hemagglutinin inhibition assay (HAI), an 80-year-old technology, and the microneutralization assay (MN). Both assays use serial dilution to provide a discrete, ranked readout of 8–14 categorical titer values for each sample. In contrast to other methods of measuring vaccine antibody levels that produce a continuous readout (i.e., mPLEX-Flu and ELISA), titering methods introduce imprecision and increase false discovery rates (FDR). In this paper, we assess the degree of such statistical errors, first with simulation studies comparing continuous data with titer data in influenza vaccine study group comparison analyses and then by analyzing actual sample data from an influenza vaccine trial. Our results show the superiority of using continuous, rather than discrete, readout assays. Compared to continuous readout assays, titering assays have a lower statistical precision and a higher FDR. The results suggested that traditional titering assays could lead to increased Type-II errors in the comparison of different therapeutic arms of an influenza vaccine trial. These statistical issues are related to the mathematical nature of titer-based assays, which we examine in detail in the simulation studies. Continuous readout assays are free of this issue, and thus it is possible that comparisons of study groups could provide different results with these two methods as we have shown in our case study.

Neovascularization and functional assessment of human pancreatic islets encapsulated with immuno‐modulatory stem cells

Background & ObjectiveDeveloping successful therapies based on re‐introduction of healthy pancreatic islets in diabetic patients requires new strategies to combat the immune response caused by implantation, as well as improving neovascularization and engraftment of the implant. Human amniotic epithelial cells (AECs) are immuno‐modulatory stem cells that can confer a local immune‐privileged environment in vivo, and block grafted tissue rejection [1]. The objective of this study was to develop a protocol for encapsulating islets with AECs, and examine the effects of encapsulation on islet function, and neovascularization.MethodsHuman pancreatic islets were individually placed in ultra‐low attachment round bottom wells with human islet medium, and were divided into three experimental groups: 1) islet‐only, 2) adipose‐derived stem cells (ASCs)+islet, and 3) AECs+islet. For groups 2 and 3, each islet was assumed to have ~2000 beta cells, and the stem cells were added to each well to encapsulate individual islets at 2:1 cell ratio. The temporal c‐peptide secretion level was measured after 3 and 6 days by perfusing the islets with basal and high glucose solutions using dynamic readout assay, and confirmed the functionality of the encapsulated islets. To assess the angiogenic properties of encapsulation, cell culture supernates were collected after 6 days of culture, and used for an angiogenesis array kit, to identify and compare the level of 52 angiogenic factors present in each group. The same test was repeated for ASC‐ and AEC‐only cultures to identify any effects caused by the cell encapsulation.Results & ConclusionsThe level of pro‐angiogenic factors increased in encapsulated groups compared to the islet‐only group (Fig. 1). A larger increase in angiogenic factors in encapsulated islets compared to stem cell‐only groups demonstrated a synergetic angiogenic effect caused by encapsulation of islets with ASCs and AECs. To test the angiogenic response in a physiological scenario, the islets were seeded on rat mesentery tissues, and cultured for 6 days, and the level of angiogenic response was compared between different groups. Our results highlight the effect of encapsulating human islets with ASCs and AECs on engraftment and neovascularization, and showcase the potential of the islet cell encapsulation method for a successful long‐term implantation in patients with diabetes.Support or Funding InformationThe Virginia Catalyst and UVA Center for Advanced BiomanufacturingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.