UMBC Chemistry & Biochemistry Department

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    Nano Moth-eye Structures Fabricated Using Ultra-thin Photoresist and Combined Dry and Wet Etches
    (SciTechnol, 2023-02-24) Sood, Rachit; Tu, Chaoran; Bamford, Douglas; Henseley, Joel; Woolf, David; Menyuk, Curtis; Singh, Narsingh; Choa, Fow-Sen
    Anti-Reflective (AR) coatings are used to suppress reflection and enhance optical transmission, but many coatings cannot withstand harsh environmental conditions. In this work, we report the fabrication of nanostructures on Gallium Arsenide (GaAs) via contact photolithography for anti-reflection applications in the mid-infrared (mid-IR) range. An E- beam mask was used to lithographically transfer nano-structure patterns to a SiO2 etching mask and then further transfer the structure to gallium arsenide wafers. With a thin layer Photo Resist (PR) along with a combination of Reactive Ion Etching (RIE) and wet Buffered Oxide Etching (BOE), we were able to transfer the nanostructure patterns from the thin PR to the thick SiO2 etching mask and then onto a wafer. The fabricated structures are squares and hexagons of feature size 900 nm, 1000 nm, 1100 nm, and the gap between two neighboring shapes is 400 nm. By varying the pitch of the structures, we observe improvement in the transmission over the mid-IR range (500-2000 cm-1 wavenumber). Experimental results of coated and uncoated GaAs are obtained using the Fourier Transform Infrared Spectroscopy (FTIR) while theoretical results of coated GaAs are shown using the Rigorous Coupled Wave Analysis (RCWA). This work provides a better success rate and a more readily available mass production technique to fabricate the sub-wavelength nanostructures. The theoretical results obtained using RCWA agree well with experimental results to show the overall 69% transmission with a one-side coated gallium arsenide wafer.
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    Effect of high energy radiation on electrical properties of synthetic bone materials
    (Wiley, 2024-02-02) Machuga, Krishna S.; Tauraso, Aria; Tauraso, Aria; Su, Ching Hua; Cullum, Brian; Arnold, Bradley; Choa, Fow-Sen; Prasad, Narasimha; Singh, Narsingh
    Hydroxyapatites have been investigated since past six decades as laser host materials. Because of their important roles in bone and teeth, these have been subjects of recent investigations. Gallium and titanium have great potential for decreasing the depletion of calcium and reducing osteoporosis. The electrical properties and polarity play important roles in regeneration of the bones. We observed growth of grains in selenium-doped gallium and titanium containing silicate hydroxyapatites. Observed morphology showed non-facetted microstructures and it helped in achieving larger grains. For the material processed for the period of longer than 70 h, we did not observe any difference in the dielectric constant and resistivity of the selenium-doped materials. For irradiating the materials, a Cs-137 γ-radiation with 5 µm curie dose was used up to 100 h. We observed that the dielectric constant and resistivity at different frequencies ranging from 100 to 100 000 Hz were affected by the high energy radiation. However, bias voltage in the range of 50–1 000 mV did no alter the dielectric constant or resistivity. This indicated that the breakdown of the material did not occur for this bias range.
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    Facile synthesis of sulfonamide libraries using a solid-polymer-supported HOBt scaffold
    (Elsevier, 2024-02-08) Guei, Jules Seh Noel; Kalivretenos, Aristotle; LaCourse, William
    Using solid-phase synthetic methods, silica-bound 1-hydroxybenzotriazole (Si-HOBt) was prepared by coupling 3-aminopropyl silica gel with 1-hydroxybenzotriazole-6-carboxylic acid (HOBt-COOH). The Si-HOBt was activated by the reaction of Si-HOBT with 3,4-dihydroxy-9,10-dioxo-anthracenesulfonyl chloride (alizarin red S chloride), resulting in the activated Si-HOBt reagent. The reagent was used to prepare a combination library of sulfonamide derivatives of selected aliphatic and aromatic amines for the development of new therapeutics.
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    Student Responses to Spaced Practice in Two Large Gateway Chemistry Courses
    (ACS, 2024-01-11) Carpenter, Tara; Hodges, Linda
    Spaced practice is a recognized effective study approach that fosters mastery of learning and retention of information. In this paper, we share one instructor’s experience in introducing a spaced practice intervention in a large general chemistry course and in encouraging students to continue the strategy in the next semester organic chemistry course. The curricular implementation spanned two years during the COVID pandemic and encompassed the instructors’ ongoing responsive efforts to enhance students’ success. Student perspectives of the perceived value and their use of spaced practice varied with the instructor’s conditions of the implementation. Offering spaced practice midway in a semester as an optional approach to homework garnered a positive student response and outcomes. However, moving to a required format for spaced practice in a subsequent semester resulted in much more mixed student feedback and outcomes. The instructor also encouraged and guided students in utilizing the strategy in the following organic chemistry course, and although over 70% of students planned to use it, only about a third actually persisted in the practice. When offering students effective study approaches through course design, instructors also have the challenge and opportunity to cultivate students’ intrinsic motivation and self-regulation, skills that enhance their success more broadly. Instructors need to consider investing time in promoting and demonstrating the impact of the method on students’ learning, nudging and encouraging students to persevere, and demonstrating metacognitive approaches to learning throughout the course to help students realize its rewards.
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    Leveraging Scientific Community Knowledge for Air Quality Model Chemistry Parameterizations
    (Air & Waste Management Association, 2024-01-24) Pye, Havala O. T.; Schwantes, Rebecca H.; Barsanti, Kelley C.; McNeill, V. Faye; Wolfe, Glenn M.
    Air pollution contributes to adverse health outcomes.Approximately 121 million people in the United States—one third of the population—live where National Ambient Air Quality Standards (NAAQS) are violated. In most cases, the criteria pollutants exceeding standards are ozone (O₃) and fine particles (PM₂.₅). In addition, 188 substances known or suspected to cause cancer or other serious health effects are designated as hazardous air pollutants (HAPs). Essentially, all O₃ and significant portions of PM₂.₅ and HAPs are produced in the atmosphere through chemical and physical processes. In the case of PM₂.₅, subcomponents formed primarily from precursor gases—sulfate, nitrate, ammonium, and secondary organic aerosol (SOA)—account for 60% of the U.S. county-level annual mean concentration. In addition, 47% of the cancer risk and 25% of the noncancer risk from HAPs have been attributed to atmospheric chemistry rather than direct emissions. In this article, we introduce the role of chemical mechanisms in air quality models, a new atmospheric science community effort, and needs for further mechanism development.
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    Structure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E bindingStructure of saguaro cactus virus 3′ translational enhancer mimics 5′ cap for eIF4E binding
    (PNAS, 2023-12-18) Ojha, Manju; Vogt, Jeff; Das, Naba Krishna; Redmond, Emily; Singh, Karndeep; Banna, Hasan Al; Sadat, Tasnia; Koirala, Deepak
    The genomes of several plant viruses contain RNA structures at their 3′ ends called cap-independent translation enhancers (CITEs) that bind the host protein factors such as mRNA 5′ cap-binding protein eIF4E for promoting cap-independent genome translation. However, the structural basis of such 5′ cap-binding protein recognition by the uncapped RNA remains largely unknown. Here, we have determined the crystal structure of a 3′ CITE, panicum mosaic virus-like translation enhancer (PTE) from the saguaro cactus virus (SCV), using a Fab crystallization chaperone. The PTE RNA folds into a three-way junction architecture with a pseudoknot between the purine-rich R domain and pyrimidine-rich Y domain, which organizes the overall structure to protrude out a specific guanine nucleotide, G18, from the R domain that comprises a major interaction site for the eIF4E binding. The superimposable crystal structures of the wild-type, G18A, G18C, and G18U mutants suggest that the PTE scaffold is preorganized with the flipped-out G18 ready to dock into the eIF4E 5′ cap-binding pocket. The binding studies with wheat and human eIF4Es using gel electrophoresis and isothermal titration calorimetry, and molecular docking computation for the PTE–eIF4E complex demonstrated that the PTE structure essentially mimics the mRNA 5′ cap for eIF4E binding. Such 5′ cap mimicry by the uncapped and structured viral RNA highlights how viruses can exploit RNA structures to mimic the host protein-binding partners and bypass the canonical mechanisms for their genome translation, providing opportunities for a better understanding of virus-host interactions and non-canonical translation mechanisms found in many pathogenic RNA viruses.
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    3D-printed quantitative microfluidics for bacteria and macrophage studies and the impact of indole on modulating itaconate in macrophages
    (2023-01-01) Kabandana, Giraso Keza Monia; Chen, Chengpeng; Chemistry & Biochemistry; Chemistry
    Microfluidics technology has advanced analytical capabilities, offering precise fluid manipulation across various scientific fields. While traditional microfluidic fabrication methods exist, 3D printing has revolutionized this field with its precision, customization, and versatility. However, the limited transparency and high costs associated with 3D printed microfluidic devices, hinder their application and widespread adoption. This dissertation addresses these challenges with innovative solutions. It introduces a groundbreaking approach by directly integrating optical components into 3D printed microfluidic systems, enabling direct optical measurements through these devices for the first time. Furthermore, a toolkit was designed to facilitate the rapid assembly of 3D printed microfluidic devices using low-cost microbore tubing, lowering the costs. These microfluidic systems were applied to the analysis of indole, a bacterial-released molecule, and nitrite, a pro-inflammatory molecule released by macrophages, frontline immune cells. Indole is released by bacteria, particularly in high concentrations during chronic infections, and it is known to suppress pro-inflammatory molecules in macrophages. Yet, the near-real-time release kinetics of indole in bacteria have remained unknown. Similarly, the near-real-time release kinetics of nitrite, a well-established pro-inflammatory molecule released by macrophages, have yet to be explored. Understanding release kinetics is essential for identifying unexpected events during time intervals and laying a foundation for therapeutic discoveries. Leveraging the newly developed systems with integrated optical devices, the release kinetics of both indole and nitrite were acquired in near-real time, with measurements taken every twenty seconds. Moreover, this dissertation investigates the impact of indole on macrophage metabolism. The hypothesis proposes that indole induces itaconate production, a metabolite known to suppress pro-inflammatory molecules. Using activated RAW 264.7 macrophages treated with various indole concentrations, the study quantified itaconate production and immune-responsive gene 1 (IRG1) levels, which is the protein that catalyzes itaconate production. The results confirmed that indole induces itaconate and unveiled a novel mechanism of indole-induced itaconate production through the aryl hydrocarbon receptor (AHR). In summary, this dissertation addresses microfluidics challenges and advances our understanding of the interplay between bacteria and macrophages. These findings hold potential for the development of new antibiotics and therapeutic interventions against chronic infections, marking a significant contribution to the scientific field.
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    A Modular, High Throughput Microfluidic Platform to Study the Effects of 3D Cell Culture on Endothelial Function
    (2023-01-01) Jones, Curtis; Chen, Chengpeng; Chemistry & Biochemistry; Chemistry
    Knowledge of the signaling effects of the microenvironment on endothelial cell function is critical for the understanding of cardiovascular disease development and progression. Currently, microfluidics is the gold standard in vitro platform for the study of endothelial cells due to the inclusion of shear stress in the model. However, these models predominantly culture cells on a traditional flat surface. To build a more complete biomimetic model, the cell culture surface should emulate the basement membrane (BM) of a blood vessel, characterized as an array of aligned microfibers. Therefore, to bridge this gap in technology, a highly customizable microfluidic system was developed with the ability to easily integrate 3D scaffolds into the model. To demonstrate the usefulness of this system, a 4.88-fold increase in nitric oxide (NO) production was observed in cells cultured on a 3D scaffold as opposed to a conventional 2D surface. This system was then modified to incorporate trans endothelial/epithelial resistance (TEER) measurements into the model, allowing for the investigation of chemical effects on barrier integrity in near real-time. Traditionally, TEER instruments are very expensive and rigid in construction, which complicates experimental design. Using an Arduino microcontroller and a pair of metal leads, a cheap customizable TEER meter was developed and implemented into the microfluidic system. Using this system, the effect of doxorubicin, a common anticancer drug, on endothelial cell barrier integrity was monitored in near real-time for a period of 24 hours. Finally, this microfluidic platform was utilized to observe a previously unknown signaling effect of surface architecture on endothelial cell function. The surface-dependent expression of beta-1 integrin was correlated to an increase of phosphorylation of the endothelial nitric oxide synthase (eNOS), which results in an increase in NO production. This microfluidic platform was shown to be a powerful tool for pre-clinical biological research and will broadly benefit the biotechnology field.
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    THE DEVELOPMENT OF DYE-DOPED SEMICONDUCTING POLYMER DOTS FOR FLUORESCENCE IMAGING AND SENSING APPLICATIONS
    (2023-01-01) Riahin, Connor; Rosenzweig, Zeev; Chemistry & Biochemistry; Chemistry
    Fluorescent semiconducting polymer dots are a new type of fluorescent nanoparticle that has shown great potential for fluorescence imaging due to their exceptional chemical and photophysical properties. Pdots are composed of semiconducting polymers that offer several advantages over inorganic QDs and organic dyes, including facile synthesis, high photostability, tunable absorption and emission, and exceptionally high absorptivity and brightness. On a per-particle basis, fluorescent Pdots are several times brighter than similar emitting QDs and organic dyes. This brightness is the result of fast emissive rates, large extinction coefficients, and high emission quantum yields. Additionally, Pdots are resistant to photobleaching, making them highly applicable to fluorescence imaging. The one major limitation of Pdots compared to organic dyes and quantum dots is their broad emission that can span several hundred nanometers. Theresearch presented in this dissertation aims to solve this problem and expand the fluorescence imaging applications of this promising nanoparticle. First, we employ a series of porphyrin dyes to tune the optical properties of Pdots. When doped into the polymer matrix, the dyes act as energy acceptors, quenching polymer emission and causing the Pdot to take on the emission properties of the dye. This significantly narrows and red-shifts Pdot fluorescence, while maintain the strong absorption properties and aqueous solubility of the Pdots. We utilized these dye-doped Pdots for NIR cellular imaging and demonstrated their potential for super-resolution fluorescence imaging. The introduction of dyes to the Pdots did produce a new problem: photobleaching. Intense energy transfer from the polymer to the dye results in rapid photobleaching of the dye and the return of polymer fluorescence. Designing dyes to be more resistant to photobleaching has not yet yielded desired results and requires further study. Second, we investigated new series of dyes to impart additional properties onto our Pdots. Using phthalocyanine dyes produced extremely bright NIR emission, though over a small range of wavelengths. BBTD allowed us to shift the wavelength of emission much deeper into the NIR, with one dye-doped Pdot reaching the NIRII window. This capability is highly desired because the NIRII window has even lower autofluorescence and scatter than NIRI, producing higher signal-to-noise ratios. Finally, we used a ruthenium dye to impart oxygen sensing properties to our Pdots, demonstrating that doped dyes maintain their sensing properties when doped into Pdots. We also found that Pdots possess excellent two-photon absorption capability, which make them interesting candidates for two-photon fluorescence microscopy. Finally. We investigated the relationship between Pdots and lipids. Amphiphilic polymers were substituted with lipids to produce Pdots with altered surface functionalization and optical properties. The interactions between Pdots and liposomes were also carried out to study their potential harmful interactions with cell membranes. We found that Pdot-lipid interactions were controlled by ligand. The distributed negative charge on PSMA prevented all interactions between the liposome and the semiconducting polymer, keeping the Pdots form disrupting the membrane. This result bodes well for PdotsÕ potential use as in vivo imaging probes, though more extensive study is needed. Our development of the dye-doped Pdot approach has led to the creation of a highly modular fluorophore system. The absorption characteristics of the nanoparticle can be altered by simply changing out the semiconducting polymer, while fluorescence properties can be tailored as desired using different organic dyes. Surface functionalization can be easily tuned by switching out ligands or with post-synthesis modification. Our Pdots have also been shown to be highly biocompatible, which makes them excellent candidates for in vivo fluorescence imaging. Once we solve photostability issues, these Pdots will be highly applicable as fluorescence sensors or detectors.
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    Banking on adaptive questions to nudge student responsibility for learning in general chemistry
    (Taylor & Francis, 2023) Carpenter, Tara; Fritz, John; Penniston, Thomas
    In this case study from the University of Maryland, Baltimore County (UMBC), we explore if and how students can be nudged to take responsibility for their learning through one of the university’s largest courses, CHEM 102 “Principles of Chemistry II.” To do so, Dr. Tara Carpenter leveraged the campus’ Blackboard learning management system (LMS) in Spring 2021, and then added the RealizeIt adaptive learning platform in Fall 2021 and Spring 2022 to implement a pedagogy of “spaced practice,” in which students have time to study, forget, reacquire, and reorganize new knowledge or content. Specifically, Carpenter leveraged large pools or “banks” of questions to guide students in their “time on task” practice and application of key concepts needed to perform well on high-stakes, summative exams. Overall, in comparing CHEM 102 final grade data between Fall 2020 and Fall 2021, we see there is not a statistically significant relationship between the treatment (i.e., course design) and reduced DFW (drop, fail, or withdraw) rates. However, if we disaggregate final grade data, we see there is an overall statistically significant increase in As (p<.01) and decrease in Cs (p <.05) and Ds (p<.05). Notably, all of this gain from increasing As appears to be from students of color (SOC), who demonstrate a nearly 4x advantage over their non-redesigned course peers in attaining this grade (p<.001), while White students demonstrated no statistically significant gain in this area. Based solely on whether students used the spaced practice environment Carpenter designed, we also see that a model predicting final grades after only 14 days into the semester is 83% accurate.
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    Perovskites for chemical and biological sensing
    Emge, Ian; Bagherzadeh, Hedyeh; Su, Ching Hua; Choa, Fow-Sen; Arnold, Bradley; Kelly, Lisa; Cullum, Brian; Mandal, K. D.; Prasad, Narasimha; Singh, Narsingh
    Objectives: The objectives was to utilize changes in polarizability/dielectric constant to determine sensing capability due to chemicals. Background: • CaCu₃Ti₄O₁₂ (CCTO) is a very good perovskite material with high dielectric constant • Literature shows dielectric value depends on processing method also • We need very high dielectric constant and high resistivity • CaCu₃Ti₄O₁₂ (CCTO). • CCTO has shown very large dielectric constant.>100000 in MHZ range. It depends on processing parameters • Very high dielectric constant may provide big changes due to chemicals
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    Sources of Formaldehyde in U.S. Oil and Gas Production Regions
    (ACS, 2023-11-13) Dix, Barbara; Li, Meng; Roosenbrand, Esther; Francoeur, Colby; Clair, Jason St.; et al
    We analyzed observational and model data to study the sources of formaldehyde over oil and gas production regions and to investigate how these observations may be used to constrain oil and gas volatile organic compound (VOC) emissions. The analysis of aircraft and satellite data consistently found that formaldehyde over oil and gas production regions during spring and summer is mostly formed by the photooxidation of precursor VOCs. Formaldehyde columns over the Permian Basin, one of the largest oil- and gas-producing regions in the United States, are correlated with the production locations. Formaldehyde simulations by the atmospheric chemistry and transport model WRF-Chem, which included oil and gas NOx and VOC emissions from the fuel-based oil and gas inventory, were in very good agreement with TROPOMI satellite measurements. Sensitivity studies illustrated that VOCs released from oil and gas activities are important precursors to formaldehyde, but other sources of VOCs contribute as well and that the formation of secondary formaldehyde is highly sensitive to NOₓ. We also investigated the ability of the chemical mechanism used in WRF-Chem to represent formaldehyde formation from oil and gas hydrocarbons by comparing against the Master Chemical Mechanism. Further, our work provides estimates of primary formaldehyde emissions from oil and gas production activities, with per basin averages ranging from 0.07 to 2.2 kg h⁻¹ in 2018. A separate estimate for natural gas flaring found that flaring emissions could contribute 5 to 12% to the total primary formaldehyde emissions for the Permian Basin in 2018.
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    Extreme Altitudes of Stratospheric Hydration by Midlatitude Convection Observed During the DCOTSS Field Campaign
    (AGU, 2023-09-26) Homeyer, Cameron R.; Smith, Jessica B.; Bedka, Kristopher M.; Bowman, Kenneth P.; Wilmouth, David M.; Ueyama, Rei; Dean-Day, Jonathan M.; Clair, Jason St.; Hannun, Reem; Hare, Jennifer; Pandey, Apoorva; Sayres, David S.; Hanisco, Thomas F.; Gordon, Andrea E.; Tinney, Emily N.
    Water vapor's contribution to Earth's radiative forcing is most sensitive to changes in its lower stratosphere concentration. One recognized pathway for rapid increases in stratospheric water vapor is tropopause-overshooting convection. Since this pathway has been rarely sampled, the NASA Dynamics and Chemistry of the Summer Stratosphere (DCOTSS) field project focused on obtaining in situ observations of stratospheric air recently affected by convection over the United States. This study reports on the extreme altitudes to which convective hydration was observed. The data show that the overworld stratosphere is routinely hydrated by convection and that past documented records of stratospheric heights of convective hydration were exceeded during several DCOTSS flights. The most extreme event sampled is highlighted, for which stratospheric water vapor was increased by up to 26% at an altitude of 19.25 km, a potential temperature of 463 K, and an ozone mixing ratio >1500 ppbv.
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    Flex-Nucleosides: A Strategic Approach to Antiviral Therapeutics
    (Springer, 2023-07-30) Seley-Radtke, Katherine; Kutz, Christianna H. M.; Thames, Joy E.
    One of the most common classes of drugs is nucleoside analogues, which have long served as a cornerstone for antiviral, antiparasitic, and anticancer treatments. This is due to their close resemblance to the naturally occurring nucleosides found in many biological processes. In terms of antiviral drug design, typically viral proteins or cellular proteins are targeted. Each has its pros and cons; however, both carry the risk of the development of drug resistance, making it imperative that novel and more effective antivirals are developed. This chapter will focus on a specific class of shaped-modified nucleoside analogues called fleximers. These flexible nucleoside analogues possess a split purine ring system, which endows flexibility to the nucleobase scaffold. This flexibility has been shown to be beneficial for increased antiviral activity but also results in broad-spectrum antiviral activity as well as the potential to overcome point mutations related to viral drug resistance. For over two decades now, the fleximer technology has been applied to numerous nucleoside analogues and has led to potent, broad-spectrum activity against a wide array of viruses including flaviviruses, filoviruses, and coronaviruses, among others. Their history and development, the various synthetic routes to realize them, and some of the biological data obtained to date for the fleximers will be discussed within this chapter.
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    Spatial Distribution Profiles of Emtricitabine, Tenofovir, Efavirenz, and Rilpivirine in Murine Tissues Following In Vivo Dosing Correlate with Their Safety Profiles in Humans
    (ACS, 2020-04-23) Seneviratne, Herana Kamal; Hamlin, Allyson N.; Heck, Carley J. S.; Bumpus, Namandjé N.
    Emtricitabine (FTC), tenofovir (TFV), efavirenz (EFV), and rilpivirine (RPV) are currently used as components of HIV combination therapy. Although these drugs are widely used in antiretroviral therapy, several organ toxicities related to TFV and EFV have been observed clinically. TFV is associated with nephrotoxicity, whereas EFV-related hepatotoxicity and neurotoxicity have been reported. While the precise molecular mechanisms related to the above-mentioned clinically observed toxicities have yet to be elucidated, understanding the local tissue distribution profiles of these drugs could yield insights into their safety profiles. To date, the distributions of these drugs in tissue following in vivo exposure are poorly understood. Therefore, in this study, we employed a matrix-assisted laser desorption/ionization mass spectrometry imaging method to generate spatial distribution profiles of FTC, TFV, EFV, and RPV in mouse tissues following in vivo dosing of following drug regimens: TFV–FTC–EFV and TFV–FTC–RPV. For this study, liver, brain, kidney, spleen, and heart tissues were obtained from mice (n = 3) following separate oral administration of the above-mentioned drug regimens. Interestingly, EFV was detected in liver, brain, and heart following TFV–FTC–EFV treatment. Additionally, hydroxylated EFV, which encompasses the cytochrome P450-dependent monooxygenated metabolites of EFV, was detected in liver, brain, spleen, and heart tissue sections. Notably, the tissue distribution profiles of RPV and hydroxylated RPV following in vivo dosing of TFV–FTC–RPV were different from EFV/hydroxylated EFV despite RPV belonging to the same drug class as EFV. In conclusion, the observed spatial distribution profiles of the study drugs are in agreement with their safety profiles in humans.
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    Twelfth-Position Deuteration of Nevirapine Reduces 12-Hydroxy-Nevirapine Formation and Nevirapine-Induced Hepatocyte Death
    (ACS, 2020-02-17) Heck, Carley J. S.; Seneviratne, Herana Kamal; Bumpus, Namandjé N.
    Cytochrome P450-dependent metabolism of the anti-HIV drug nevirapine (NVP) to 12-hydroxy-NVP (12-OHNVP) has been implicated in NVP toxicities. We investigated the impact of twelfth-position trideuteration (12-D3NVP) on the hepatic metabolism of and response to NVP. Formation of 12-OHNVP decreased in human (10.6-fold) and mouse (4.6-fold) hepatocytes incubated with 10 μM 12-D3NVP vs NVP. An observed kinetic isotope effect of 10.1 was measured in human liver microsomes. During mouse hepatocyte treatment (400 μM) with NVP or 12-D3NVP, cell death was reduced 30% with 12-D3NVP vs NVP, while glucuronidated and glutathione-conjugated metabolites increased with 12-D3NVP vs NVP. Using mass spectrometry proteomics, changes in hepatocyte protein expression, including an increase in stress marker insulin-like growth factor-binding protein 1 (IGFBP-1), were observed with 12-D3NVP vs NVP. These results demonstrate that while deuteration can reduce P450 metabolite formation, impacts on phase II metabolism and hepatocyte protein expression should be considered when employing deuteration to reduce P450 metabolite-related hepatotoxicity.
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    Identification of Novel UGT1A1 Variants Including UGT1A1 454C>A through the Genotyping of Healthy Participants of the HPTN 077 Study
    (ACS, 2021-01-21) Seneviratne, Herana Kamal; Hamlin, Allyson N.; Li, Sue; Grinsztejn, Beatriz; Dawood, Halima; Liu, Albert Y.; Kuo, Irene; Hosseinipour, Mina C.; Panchia, Ravindre; Cottle, Leslie; Chau, Gordon; Adeyeye, Adeola; Rinehart, Alex R.; McCauley, Marybeth; Eron, Joseph S.; Cohen, Myron S.; Landovitz, Raphael J.; Hendrix, Craig W.; Bumpus, Namandjé N.
    Cabotegravir (CAB) is an integrase strand-transfer inhibitor of HIV that has proven effective for HIV treatment and prevention in a long-acting injectable formulation, typically preceded by an oral formulation lead-in phase. Previous in vitro studies have demonstrated that CAB is primarily metabolized via glucuronidation by uridine diphosphate glucuronosyltransferase (UGT) 1A1 and 1A9. In this study, we performed next-generation sequencing of genomic DNA isolated from the HPTN 077 participants to explore the variants within UGT1A1 and UGT1A9. Additionally, to enable correlation of UGT1A1 and UGT1A9 genotypes with plasma CAB-glucuronide levels, we quantified glucuronidated CAB following both oral administration of CAB and intramuscular injection of long-acting CAB. From these studies, 48 previously unreported variants of UGT1A1 and UGT1A9 were detected. Notably, 5/68 individuals carried a UGT1A1 454C>A variant that resulted in amino acid substitution P152T, and the use of in silico tools predicted a deleterious effect of the P152T substitution. Thus, the impact of this mutant on a range of UGT1A1 substrates was tested using a COS-7 cell-based assay. The glucuronide conjugates of CAB, dolutegravir, and raltegravir, were not formed in the COS-7 cells expressing the UGT1A1 P152T mutant. Further, formation of glucuronides of raloxifene and 7-ethyl-10-hydroxycamptothecin were reduced in the cells expressing the UGT1A1 P152T mutant. Using the same approach, we tested the activities of two UGT1A9 mutants, UGT1A9 H217Y and UGT1A9 R464G, and found that these mutations were tolerated and decreased function, respectively. These data provide insight into previously unreported genetic variants of UGT1A1 and UGT1A9.
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    Atp7b-dependent choroid plexus dysfunction causes transient copper deficit and metabolic changes in the developing mouse brain
    (PLOS, 2023-01-20) Washington-Hughes, Clorissa L.; Roy, Shubhrajit; Seneviratne, Herana Kamal; Karuppagounder, Senthilkumar S.; Morel, Yulemni; Jones, Jace W.; Zak, Alex; Xiao, Tong; Boronina, Tatiana N.; Cole, Robert N.; Bumpus, Namandjé N.; Chang, Christopher J.; Dawson, Ted M.; Lutsenko, Svetlana
    Copper (Cu) has a multifaceted role in brain development, function, and metabolism. Two homologous Cu transporters, Atp7a (Menkes disease protein) and Atp7b (Wilson disease protein), maintain Cu homeostasis in the tissue. Atp7a mediates Cu entry into the brain and activates Cu-dependent enzymes, whereas the role of Atp7b is less clear. We show that during postnatal development Atp7b is necessary for normal morphology and function of choroid plexus (ChPl). Inactivation of Atp7b causes reorganization of ChPl’ cytoskeleton and cell-cell contacts, loss of Slc31a1 from the apical membrane, and a decrease in the length and number of microvilli and cilia. In ChPl lacking Atp7b, Atp7a is upregulated but remains intracellular, which limits Cu transport into the brain and results in significant Cu deficit, which is reversed only in older animals. Cu deficiency is associated with down-regulation of Atp7a in locus coeruleus and catecholamine imbalance, despite normal expression of dopamine-β-hydroxylase. In addition, there are notable changes in the brain lipidome, which can be attributed to inhibition of diacylglyceride-to-phosphatidylethanolamine conversion. These results identify the new role for Atp7b in developing brain and identify metabolic changes that could be exacerbated by Cu chelation therapy.
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    Achieving a Deeper Understanding of Drug Metabolism and Responses Using Single-Cell Technologies
    (ASPET, 2023-03-01) Wheeler, Abigail M.; Eberhard, Colten D.; Mosher, Eric P.; Yuan, Yuting; Wilkins, Hannah N.; Seneviratne, Herana Kamal; Orsburn, Benjamin C.; Bumpus, Namandje N.
    Recent advancements in single-cell technologies have enabled detection of RNA, proteins, metabolites, and xenobiotics in individual cells, and the application of these technologies has the potential to transform pharmacological research. Single-cell data has already resulted in the development of human and model species cell atlases, identifying different cell types within a tissue, further facilitating the characterization of tumor heterogeneity, and providing insight into treatment resistance. Research discussed in this review demonstrates that distinct cell populations express drug metabolizing enzymes to different extents, indicating there may be variability in drug metabolism not only between organs, but within tissue types. Additionally, we put forth the concept that single-cell analyses can be used to expose underlying variability in cellular response to drugs, providing a unique examination of drug efficacy, toxicity, and metabolism. We will outline several of these techniques: single-cell RNA-sequencing and mass cytometry to characterize and distinguish different cell types, single-cell proteomics to quantify drug metabolizing enzymes and characterize cellular responses to drug, capillary electrophoresis-ultrasensitive laser-induced fluorescence detection and single-probe single-cell mass spectrometry for detection of drugs, and others. Emerging single-cell technologies such as these can comprehensively characterize heterogeneity in both cell-type-specific drug metabolism and response to treatment, enhancing progress toward personalized and precision medicine. SIGNIFICANCE STATEMENT Recent technological advances have enabled the analysis of gene expression and protein levels in single cells. These types of analyses are important to investigating mechanisms that cannot be elucidated on a bulk level, primarily due to the variability of cell populations within biological systems. Here, we summarize cell-type-specific drug metabolism and how pharmacologists can utilize single-cell approaches to obtain a comprehensive understanding of drug metabolism and cellular heterogeneity in response to drugs.