IU Indianapolis ScholarWorks :: Browsing by Author "Siegel, Amanda P."

Browsing by Author "Siegel, Amanda P."

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Canine-Inspired Chemometric Analysis of Volatile Organic Compounds in Urine Headspace to Distinguish Prostate Cancer in Mice and Men
(MDPI, 2023-02-20) Woollam, Mark; Siegel, Amanda P.; Munshi, Adam; Liu, Shengzhi; Tholpady, Sunil; Gardner, Thomas; Li, Bai-Yan; Yokota, Hiroki; Agarwal, Mangilal; Chemistry and Chemical Biology, School of Science
Canines can identify prostate cancer with high accuracy by smelling volatile organic compounds (VOCs) in urine. Previous studies have identified VOC biomarkers for prostate cancer utilizing solid phase microextraction (SPME) gas chromatography-mass spectrometry (GC-MS) but have not assessed the ability of VOCs to distinguish aggressive cancers. Additionally, previous investigations have utilized murine models to identify biomarkers but have not determined if the results are translatable to humans. To address these challenges, urine was collected from mice with prostate cancer and men undergoing prostate cancer biopsy and VOCs were analyzed by SPME GC-MS. Prior to analysis, SPME fibers/arrows were compared, and the fibers had enhanced sensitivity toward VOCs with a low molecular weight. The analysis of mouse urine demonstrated that VOCs could distinguish tumor-bearing mice with 100% accuracy. Linear discriminant analysis of six VOCs in human urine distinguished prostate cancer with sensitivity = 75% and specificity = 69%. Another panel of seven VOCs could classify aggressive cancer with sensitivity = 78% and specificity = 85%. These results show that VOCs have moderate accuracy in detecting prostate cancer and a superior ability to stratify aggressive tumors. Furthermore, the overlap in the structure of VOCs identified in humans and mice shows the merit of murine models for identifying biomarker candidates.
Chemometric Analysis of Urinary Volatile Organic Compounds to Monitor the Efficacy of Pitavastatin Treatments on Mammary Tumor Progression over Time
(MDPI, 2022-07) Grocki, Paul; Woollam, Mark; Wang, Luqi; Liu, Shengzhi; Kalra, Maitri; Siegel, Amanda P.; Li, Bai-Yan; Yokota, Hiroki; Agarwal, Mangilal; Chemistry and Chemical Biology, School of Science
Volatile organic compounds (VOCs) in urine are potential biomarkers of breast cancer. Previously, our group has investigated breast cancer through analysis of VOCs in mouse urine and identified a panel of VOCs with the ability to monitor tumor progression. However, an unanswered question is whether VOCs can be exploited similarly to monitor the efficacy of antitumor treatments over time. Herein, subsets of tumor-bearing mice were treated with pitavastatin at high (8 mg/kg) and low (4 mg/kg) concentrations, and urine was analyzed through solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS). Previous investigations using X-ray and micro-CT analysis indicated pitavastatin administered at 8 mg/kg had a protective effect against mammary tumors, whereas 4 mg/kg treatments did not inhibit tumor-induced damage. VOCs from mice treated with pitavastatin were compared to the previously analyzed healthy controls and tumor-bearing mice using chemometric analyses, which revealed that mice treated with pitavastatin at high concentrations were significantly different than tumor-bearing untreated mice in the direction of healthy controls. Mice treated with low concentrations demonstrated significant differences relative to healthy controls and were reflective of tumor-bearing untreated mice. These results show that urinary VOCs can accurately and noninvasively predict the efficacy of pitavastatin treatments over time.
Cholesterol-Induced Buckling in Physisorbed Polymer-Tethered Lipid Monolayers
(MDPI, 2013-04-06) Hussain, Noor F.; Siegel, Amanda P.; Johnson, Merrell A.; Naumann, Christoph A.; Chemistry and Chemical Biology, School of Science
The influence of cholesterol concentration on the formation of buckling structures is studied in a physisorbed polymer-tethered lipid monolayer system using epifluorescence microscopy (EPI) and atomic force microscopy (AFM). The monolayer system, built using the Langmuir-Blodgett (LB) technique, consists of 3 mol % poly(ethylene glycol) (PEG) lipopolymers and various concentrations of the phospholipid, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and cholesterol (CHOL). In the absence of CHOL, AFM micrographs show only occasional buckling structures, which is caused by the presence of the lipopolymers in the monolayer. In contrast, a gradual increase of CHOL concentration in the range of 0–40 mol % leads to fascinating film stress relaxation phenomena in the form of enhanced membrane buckling. Buckling structures are moderately deficient in CHOL, but do not cause any notable phospholipid-lipopolymer phase separation. Our experiments demonstrate that membrane buckling in physisorbed polymer-tethered membranes can be controlled through CHOL-mediated adjustment of membrane elastic properties. They further show that CHOL may have a notable impact on molecular confinement in the presence of crowding agents, such as lipopolymers. Our results are significant, because they offer an intriguing prospective on the role of CHOL on the material properties in complex membrane architecture.
Detection of Volatile Organic Compounds (VOCs) in Urine via Gas Chromatography-Mass Spectrometry QTOF to Differentiate Between Localized and Metastatic Models of Breast Cancer
(Springer Nature, 2019-02-21) Woollam, Mark; Teli, Meghana; Angarita-Rivera, Paula; Liu, Shengzhi; Siegel, Amanda P.; Yokota, Hiroki; Agarwal, Mangilal; Chemistry and Chemical Biology, School of Science
Breast cancer is the most common cancer detected in women and current screening methods for the disease are not sensitive. Volatile organic compounds (VOCs) include endogenous metabolites that provide information about health and disease which might be useful to develop a better screening method for breast cancer. The goal of this study was to classify mice with and without tumors and compare tumors localized to the mammary pad and tumor cells injected into the iliac artery by differences in VOCs in urine. After 4T1.2 tumor cells were injected into BALB/c mice either in the mammary pad or into the iliac artery, urine was collected, VOCs from urine headspace were concentrated by solid phase microextraction and results were analyzed by gas chromatography-mass spectrometry quadrupole time-of-flight. Multivariate and univariate statistical analyses were employed to find potential biomarkers for breast cancer and metastatic breast cancer in mice models. A set of six VOCs classified mice with and without tumors with an area under the receiver operator characteristic (ROC AUC) of 0.98 (95% confidence interval [0.85, 1.00]) via five-fold cross validation. Classification of mice with tumors in the mammary pad and iliac artery was executed utilizing a different set of six VOCs, with a ROC AUC of 0.96 (95% confidence interval [0.75, 1.00]).
Engineering the electrospinning of MWCNTs/epoxy nanofiber scaffolds to enhance physical and mechanical properties of CFRPs
(Elsevier, 2021-09) Wable, Vidya; Biswas, Pias Kumar; Moheimani, Reza; Aliahmad, Nojan; Omole, Peter; Siegel, Amanda P.; Agarwal, Mangilal; Dalir, Hamid; Mechanical Engineering, School of Engineering and Technology
A cost-effective approach to improve the physical and mechanical properties of carbon fiber reinforced polymer (CFRP) prepreg composites, where electrospun multiwalled carbon nanotubes (MWCNTs)/epoxy nanofibers were synthesized and incorporated in between the layers of conventional CFRP prepreg composite has been presented. MWCNT-aligned epoxy nanofibers were successfully produced by an optimized electrospinning process. Nanofibers were deposited directly onto prepreg layers to achieve improved adhesion and interfacial bonding, leading to added strength and improvements in other mechanical properties. Thus, interlaminar shear strength (ILSS) and fatigue performance at high-stress regimes increased by 29% and 27%, respectively. Barely visible impact damage (BVID) energy increased significantly by up to 45%. The thermal and electrical conductivities were also enhanced significantly due to the presence of the highly conductive MWCNT networks between the CFRP layers. The presented method was capable of uniformly depositing high contents of MWCNTs at interlaminar ply interface of prepregs to strengthen/enhance CFRP properties, which has not been previously shown to be possible due to high resin viscosity caused by randomly oriented MWCNTs in epoxy system.
Exosome-Mediated Crosstalk between Keratinocytes and Macrophages in Cutaneous Wound Healing
(ACS, 2020-09) Zhou, Xiaoju; Brown, Brooke A.; Siegel, Amanda P.; El Masry, Mohamed S.; Zeng, Xuyao; Song, Woran; Das, Amitava; Khandelwal, Puneet; Clark, Andrew; Singh, Kanhaiya; Guda, Poornachander R.; Gorain, Mahadeo; Timsina, Lava; Xuan, Yi; Jacobson, Stephen C.; Novotny, Milos V.; Roy, Sashwati; Agarwal, Mangilal; Lee, Robert J.; Sen, Chandan K.; Clemmer, David E.; Ghatak, Subhadip; Surgery, School of Medicine
Bidirectional cell–cell communication involving exosome-borne cargo such as miRNA has emerged as a critical mechanism for wound healing. Unlike other shedding vesicles, exosomes selectively package miRNA by SUMOylation of heterogeneous nuclear ribonucleoproteinA2B1 (hnRNPA2B1). In this work, we elucidate the significance of exosome in keratinocyte–macrophage crosstalk following injury. Keratinocyte-derived exosomes were genetically labeled with GFP-reporter (Exoκ-GFP) using tissue nanotransfection (TNT), and they were isolated from dorsal murine skin and wound-edge tissue by affinity selection using magnetic beads. Surface N-glycans of Exoκ-GFP were also characterized. Unlike skin exosome, wound-edge Exoκ-GFP demonstrated characteristic N-glycan ions with abundance of low-base-pair RNA and was selectively engulfed by wound macrophages (ωmϕ) in granulation tissue. In vitro addition of wound-edge Exoκ-GFP to proinflammatory ωmϕ resulted in conversion to a proresolution phenotype. To selectively inhibit miRNA packaging within Exoκ-GFPin vivo, pH-responsive keratinocyte-targeted siRNA-hnRNPA2B1 functionalized lipid nanoparticles (TLNPκ) were designed with 94.3% encapsulation efficiency. Application of TLNPκ/si-hnRNPA2B1 to the murine dorsal wound-edge significantly inhibited expression of hnRNPA2B1 by 80% in epidermis compared to the TLNPκ/si-control group. Although no significant difference in wound closure or re-epithelialization was observed, the TLNPκ/si-hnRNPA2B1 treated group showed a significant increase in ωmϕ displaying proinflammatory markers in the granulation tissue at day 10 post-wounding compared to the TLNPκ/si-control group. Furthermore, TLNPκ/si-hnRNPA2B1 treated mice showed impaired barrier function with diminished expression of epithelial junctional proteins, lending credence to the notion that unresolved inflammation results in leaky skin. This work provides insight wherein Exoκ-GFP is recognized as a major contributor that regulates macrophage trafficking and epithelial barrier properties postinjury.
Gaussian Process Regression and Monte Carlo Simulation to Determine VOC Biomarker Concentrations Via Chemiresistive Gas Nanosensors
(IEEE Xplore, 2021-06) Rivera, Paula Angarita; Woollam, Mark; Siegel, Amanda P.; Agarwal, Mangilal; Mechanical and Energy Engineering, School of Engineering and Technology
Utilizing chemiresistive gas sensors for volatile organic compound (VOC) detection has been a growing area of investigation in the last decade. VOCs have been extensively studied as potential biomarkers for biomedical applications as they are byproducts of metabolic pathways which are dysregulated by disease. Therefore, sensor arrays have been fabricated in previous studies to detect VOC biomarkers. In the process of testing these sensors, it is highly advantageous to quantify the concentration of the VOC biomarkers with high accuracy to diagnose the disease with high sensitivity and specificity. To investigate, analyze, and understand the relation between the concentrations of the VOC to the sensor resistance response, Gaussian Process (GP) models were implemented to predict the behavior of the data with respect to the resistance when the sensor is exposed to a range of concentrations of VOCs. Additionally, the relation between the concentration and resistance of the sensor was studied to predict the concentration of the VOC when a resistance is obtained. Monte Carlo Simulation Sampling from the GP model was utilized to generate data to further understand the trend. The results demonstrated that the relation between the concentration and resistance is linear. The model was tested with sampling data and its accuracy was evaluated.
Inferring diffusion dynamics from FCS in heterogeneous nuclear environments
(Elsevier, 2015-07-07) Tsekouras, Konstantinos; Siegel, Amanda P.; Day, Richard N.; Pressé, Steve; Department of Physics, School of Science
Fluorescence correlation spectroscopy (FCS) is a noninvasive technique that probes the diffusion dynamics of proteins down to single-molecule sensitivity in living cells. Critical mechanistic insight is often drawn from FCS experiments by fitting the resulting time-intensity correlation function, G(t), to known diffusion models. When simple models fail, the complex diffusion dynamics of proteins within heterogeneous cellular environments can be fit to anomalous diffusion models with adjustable anomalous exponents. Here, we take a different approach. We use the maximum entropy method to show-first using synthetic data-that a model for proteins diffusing while stochastically binding/unbinding to various affinity sites in living cells gives rise to a G(t) that could otherwise be equally well fit using anomalous diffusion models. We explain the mechanistic insight derived from our method. In particular, using real FCS data, we describe how the effects of cell crowding and binding to affinity sites manifest themselves in the behavior of G(t). Our focus is on the diffusive behavior of an engineered protein in 1) the heterochromatin region of the cell's nucleus as well as 2) in the cell's cytoplasm and 3) in solution. The protein consists of the basic region-leucine zipper (BZip) domain of the CCAAT/enhancer-binding protein (C/EBP) fused to fluorescent proteins.
Influence of Choline Chloride/Urea and Glycerol Plasticizers on the Mechanical Properties of Thermoplastic Starch Plastics
(MDPI, 2024-03-09) Staker, Jacob; Schott, Sydney; Singh, Riya; Collier, Kourtney; Druschel, Gregory; Siegel, Amanda P.; Tovar, Andres; Chemistry and Chemical Biology, School of Science
Bio-based plastics made of food-safe compostable materials, such as thermoplastic starch (TPS), can be designed into films that have potential to replace many non-biodegradable single-use plastic (SUP) items. TPS film characteristics, such as elongation at break and tensile strength, are largely affected by the choice of the plasticizers used in formulation. Our work identifies the mechanical properties and the chemical structural differences between TPS films made with two different plasticizer mixtures that have not yet been compared alongside one another: deep eutectic solvent choline chloride/urea (1:2) (CC:U) and glycerol with an acetic acid catalyst (AA:G). Potato-based TPS samples were formed by mixing each plasticizer with a consistent amount of potato starch and distilled water with heat. After gelation formation, the viscous TPS mixture was centrifuged to degas and extruded. Films were dried at controlled room temperature. Characterization included the tensile testing of coupons according to ASTM (American Society of Testing and Materials) standard D638, attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray diffraction (XRD), melting point (MP), and scanning electron microscopy (SEM). The AA:G films displayed significantly higher tensile strength (M = 2.04 ± 1.24 MPa) than the CC:U films (M = 0.18 ± 0.08 MPa); however, the CC:U films had higher elongation at break (M = 47.2 ± 3.6%) than the AA:G films (M = 31.1 ± 12.6%). This can be explained by the difference in functional groups, composition, and the degree of crystallinity evidenced by the FTIR, XRD, MP, and SEM results. Our findings suggest that potato-based TPS films with an AA:G plasticizer mixture hold promise for SUP applications that require more strength, while CC:U films may be more suited for wraps and bags that require flexibility. These innovations can aid to mitigate the environmental impact of harmful plastic waste.
Ligand binding alters dimerization and sequestering of urokinase receptors in raft-mimicking lipid mixtures
(Elsevier, 2014-11-04) Ge, Yifan; Siegel, Amanda P.; Jordan, Rainer; Naumann, Christoph A.; Department of Chemistry & Chemical Biology, School of Science
Lipid heterogeneities, such as lipid rafts, are widely considered to be important for the sequestering of membrane proteins in plasma membranes, thereby influencing membrane protein functionality. However, the underlying mechanisms of such sequestration processes remain elusive, in part, due to the small size and often transient nature of these functional membrane heterogeneities in cellular membranes. To overcome these challenges, here we report the sequestration behavior of urokinase receptor (uPAR), a glycosylphosphatidylinositol-anchored protein, in a planar model membrane platform with raft-mimicking lipid mixtures of well-defined compositions using a powerful optical imaging platform consisting of confocal spectroscopy XY-scans, photon counting histogram, and fluorescence correlation spectroscopy analyses. This methodology provides parallel information about receptor sequestration, oligomerization state, and lateral mobility with single molecule sensitivity. Most notably, our experiments demonstrate that moderate changes in uPAR sequestration are not only associated with modifications in uPAR dimerization levels, but may also be linked to ligand-mediated allosteric changes of these membrane receptors. Our data show that these modifications in uPAR sequestration can be induced by exposure to specific ligands (urokinase plasminogen activator, vitronectin), but not via adjustment of the cholesterol level in the planar model membrane system. Good agreement of our key findings with published results on cell membranes confirms the validity of our model membrane approach. We hypothesize that the observed mechanism of receptor translocation in the presence of raft-mimicking lipid mixtures is also applicable to other glycosylphosphatidylinositol-anchored proteins.

Browsing by Author "Siegel, Amanda P."

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