OU - Faculty and Staff Publications

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Open Access
Building Natural Product Libraries Using Quantitative Clade-Based and Chemical Clustering Strategies
(2021-10-26) Anderson, Victoria M.; Wendt, Karen L.; Najar, Fares Z.; McCall, Laura-Isobel; Cichewicz, Robert H.
The success of natural product-based drug discovery is predicated on having chemical collections that offer broad coverage of metabolite diversity. We propose a simple set of tools combining genetic barcoding and metabolomics to help investigators build natural product libraries aimed at achieving predetermined levels of chemical coverage. It was found that such tools aided in identifying overlooked pockets of chemical diversity within taxa, which could be useful for refocusing collection strategies. We have used fungal isolates identified as Alternaria from a citizen-science-based soil collection to demonstrate the application of these tools for assessing and carrying out predictive measurements of chemical diversity in a natural product collection. Within Alternaria, different subclades were found to contain nonequivalent levels of chemical diversity. It was also determined that a surprisingly modest number of isolates (195 isolates) was sufficient to afford nearly 99% of Alternaria chemical features in the data set. However, this result must be considered in the context that 17.9% of chemical features appeared in single isolates, suggesting that fungi like Alternaria might be engaged in an ongoing process of actively exploring nature’s metabolic landscape. Our results demonstrate that combining modest investments in securing internal transcribed spacer (ITS)-based sequence information (i.e., establishing gene-based clades) with data from liquid chromatography-mass spectrometry (i.e., generating feature accumulation curves) offers a useful route to obtaining actionable insights into chemical diversity coverage trends in a natural product library. It is anticipated that these outcomes could be used to improve opportunities for accessing bioactive molecules that serve as the cornerstone of natural product-based drug discovery.
Open Access
Capitalizing resolving power of density gradient ultracentrifugation by freezing and precisely slicing centrifuged solution : enabling identification of complex proteins from mitochondria by matrix assisted laser desorption/ionization time-of-flight mass spectrometry
(2016) Yu, Haiqing; Lu, Joann J.; Rao, Wei; Liu, Shaorong
Density gradient centrifugation is widely utilized for various high purity sample preparations, and density gradient ultracentrifugation (DGU) is often used for more resolution-demanding purification of organelles and protein complexes. Accurately locating different isopycnic layers and precisely extracting solutions from these layers play a critical role in achieving high-resolution DGU separations. In this technique note, we develop a DGU procedure by freezing the solution rapidly (but gently) after centrifugation to fix the resolved layers and by slicing the frozen solution to fractionate the sample. Because the thickness of each slice can be controlled to be as thin as 10 micrometers, we retain virtually all the resolution produced by DGU. To demonstrate the effectiveness of this method, we fractionate complex V from HeLa mitochondria using a conventional technique and this freezing-slicing (F-S) method. The comparison indicates that our F-S method can reduce complex V layer thicknesses by ~40%. After fractionation, we analyze complex V proteins directly on a matrix assisted laser desorption/ionization, time-of-flight mass spectrometer. Twelve out of fifteen subunits of complex V are positively identified. Our method provides a practical protocol to identify proteins from complexes, which is useful to investigate biomolecular complexes and pathways in various conditions and cell types.
Open Access
Kinetically Doped Silica Sol–Gel Optical Biosensors: Expanding Potential Through Dip-Coating
(2018-07-17) Crosley, Matthew S.; Yip, Wai Tak
Kinetic doping has previously been shown to be an effective method of doping silica sol−gel thin films with an enzyme to construct biosensors. Until now, kinetic doping has only been applied to films produced through the spincoating method. In this study, we present the use of dipcoating to produce thin films kinetically doped for biosensor development. In this way, kinetically doped biosensors may benefit from the increased range of substrate material shapes and sizes that may be easily coated through dip-coating but not spin-coating. The biosensors produced through dipcoating continue to show enhanced performance over more conventional enzyme loading methods with horseradish peroxidase and cytochrome C samples, showing an increase of 2400× and 1300× in enzyme concentration over that in their loading solutions, respectively. These correspond to enzyme concentrations of 5.37 and 10.57 mmol/L all while preserving a modest catalytic activity for the detection of hydrogen peroxide by horseradish peroxidase. This leads to a 77% and 88% increase in the total amount of horseradish peroxidase and cytochrome C, respectively, over that from coating the same glass coverslip via spincoating methods.
Open Access
Local Phenomena Shape Backyard Soil Metabolite Composition
(2020-02-29) Nguyen, Tra D.; Lesani, Mahbobeh; Forrest, Ines; Lan, Yunpeng; Dean, Danya A.; Gibaut, Quentin M. R.; Guo, Yanting; Hossain, Ekram; Olvera, Marcela; Panlilio, Hannah; Parab, Adwaita R.; Wu, Chaoyi; Bernatchez, Jean A.; Cichewicz, Robert H.; McCall, Laura-Isobel
Soil covers most of Earth’s continental surface and is fundamental to life-sustaining processes such as agriculture. Given its rich biodiversity, soil is also a major source for natural product drug discovery from soil microorganisms. However, the study of the soil small molecule profile has been challenging due to the complexity and heterogeneity of this matrix. In this study, we implemented high-resolution liquid chromatography–tandem mass spectrometry and large-scale data analysis tools such as molecular networking to characterize the relative contributions of city, state and regional processes on backyard soil metabolite composition, in 188 soil samples collected from 14 USA States, representing five USA climate regions. We observed that region, state and city of collection all influence the overall soil metabolite profile. However, many metabolites were only detected in unique sites, indicating that uniquely local phenomena also influence the backyard soil environment, with both human-derived and naturally-produced (plant-derived, microbially-derived) metabolites identified. Overall, these findings are helping to define the processes that shape the backyard soil metabolite composition, while also highlighting the need for expanded metabolomic studies of this complex environment.
Open Access
Mapping of host-parasite-microbiome interactions reveals metabolic determinants of tropism and tolerance in Chagas disease
(2020-07-22) Hossain, Ekram; Khanam, Sharmily; Dean, Danya; Wu, Si; Lostracco-Johnson, Sharon; Thomas, D.; Kane, Shelley; Parab, Adwaita; Flores, Karina; Katemauswa, Mitchelle; Gosmanov, Camil; Hayes, Stephanie E.; Zhang, Yiming; Danyang, Li; Woelfel-Monsivais, Christine; Sankaranarayanan, Krithivasan; McCall, Laura-Isobel
Chagas disease (CD) is a parasitic disease caused by Trypanosoma cruzi protozoa, presenting with cardiomyopathy, megaesophagus, and/or megacolon. To determine the mechanisms of gastrointestinal (GI) CD tissue tropism, we systematically characterized the spatial localization of infection-induced metabolic and microbiome alterations, in a mouse model of CD. Notably, the impact of the transition between acute and persistent infection differed between tissue sites, with sustained large-scale effects of infection in the esophagus and large intestine, providing a potential mechanism for the tropism of CD within the GI tract. Infection affected acylcarnitine metabolism; carnitine supplementation prevented acute-stage CD mortality without affecting parasite burden by mitigating infection-induced metabolic disturbances and reducing cardiac strain. Overall, results identified a previously-unknown mechanism of disease tolerance in CD, with potential for new therapeutic regimen development. More broadly, results highlight the potential of spatially resolved metabolomics to provide insight into disease pathogenesis and infectious disease drug development.
Open Access
(NH4)2AgX3 (X = Br, I): 1D Silver Halides with Broadband White Light Emission and Improved Stability
(2021-06-17) Creason, Tielyr D.; Fattal, Hadiah; Gilley, Isaiah W.; McWhorter, Timothy M.; Du, Mao-Hua; Saparov, Bayram
Recently, ternary copper(I) halides have emerged as alternatives to lead halide perovskites for light emission applications. Despite their high-efficiency photoluminescence (PL) properties, most copper(I) halides are blue emitters with unusually poor tunability of their PL properties. Here, we report the impact of substitution of copper with silver in the high-efficiency blue-emitting Cu(I) halides through hydrothermal synthesis and characterization of (NH4)2AgX3 (X = Br, I). (NH4)2AgX3 are found to exhibit contrasting light emission properties compared to the blue-emitting Cu(I) analogues. Thus, (NH4)2AgBr3 and (NH4)2AgI3 exhibit broadband whitish light emission at room temperature with PL maxima at 394 and 534 nm and full width at half-maximum values of 142 and 114 nm, respectively. Based on our combined experimental and computational results, the broadband emission in (NH4)2AgX3 is attributed to the presence of high-stability self-trapped excitons and defect-bound excitons. (NH4)2AgBr3 and (NH4)2AgI3 both have significantly improved air and moisture stability as compared to the related copper(I) halides, which are prone to degradation via oxidation. Our results suggest that silver halides should be considered alongside their copper analogues for high-efficiency light emission applications.
Open Access
Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes
(2016-11-21) Motley, Jeremy L.; Stamps, Blake W.; Mitchell, Carter A.; Thompson, Alec T.; Cross, Jayson; You, Jianlan; Powell, Douglas R.; Stevenson, Bradley S.; Cichewicz, Robert H.
Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.
Open Access
PEGylation of Polyethylenimine Lowers Acute Toxicity while Retaining Anti-Biofilm and β-Lactam Potentiation Properties against Antibiotic-Resistant Pathogens
(2020-09-29) Lam, Anh K.; Moen, Erika L.; Pusavat, Jennifer; Wouters, Cassandra L.; Panlilio, Hannah; Ferrell, Maya J.; Houck, Matthew B.; Glatzhofer, Daniel T.; Rice, Charles V.
Bacterial biofilms, often impenetrable to antibiotic medications, are a leading cause of poor wound healing. The prognosis is worse for wounds with biofilms of antimicrobial-resistant (AMR) bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant S. epidermidis (MRSE), and multi-drug resistant Pseudomonas aeruginosa (MDR-PA). Resistance hinders initial treatment of standard-of-care antibiotics. The persistence of MRSA, MRSE, and/or MDR-PA often allows acute infections to become chronic wound infections. The water-soluble hydrophilic properties of low-molecular-weight (600 Da) branched polyethylenimine (600 Da BPEI) enable easy drug delivery to directly attack AMR and biofilms in the wound environment as a topical agent for wound treatment. To mitigate toxicity issues, we have modified 600 Da BPEI with polyethylene glycol (PEG) in a straightforward one-step reaction. The PEG–BPEI molecules disable β-lactam resistance in MRSA, MRSE, and MDR-PA while also having the ability to dissolve established biofilms. PEG-BPEI accomplishes these tasks independently, resulting in a multifunction potentiation agent. We envision wound treatment with antibiotics given topically, orally, or intravenously in which external application of PEG–BPEIs disables biofilms and resistance mechanisms. In the absence of a robust pipeline of new drugs, existing drugs and regimens must be re-evaluated as combination(s) with potentiators. The PEGylation of 600 Da BPEI provides new opportunities to meet this goal with a single compound whose multifunction properties are retained while lowering acute toxicity.
Open Access
Perspectives on Viral RNA Genomes and the RNA Folding Problem
(2020-10-05) Schroeder, Susan J.
Viral RNA genomes change shape as virus particles disassemble, form replication complexes, attach to ribosomes for translation, evade host defense mechanisms, and assemble new virus particles. These structurally dynamic RNA shapeshifters present a challenging RNA folding problem, because the RNA sequence adopts multiple structures and may sometimes contain regions of partial disorder. Recent advances in high resolution asymmetric cryoelectron microscopy and chemical probing provide new ways to probe the degree of structure and disorder, and have identified more than one conformation in dynamic equilibrium in viral RNA. Chemical probing and the Detection of RNA Folding Ensembles using Expectation Maximization (DREEM) algorithm has been applied to studies of the dynamic equilibrium conformations in HIV RNA in vitro, in virio, and in vivo. This new type of data provides insight into important questions about virus assembly mechanisms and the fundamental physical forces driving virus particle assembly.
Open Access
Representation of the QM Subsystem for Long-Range Electrostatic Interaction in Non-Periodic Ab Initio QM/MM Calculations
(2018-09-29) Pan, Xaoliang; Rosta, Edina; Shao, Yihan
In QM/MM calculations, it is essential to handle electrostatic interactions between the QM and MM subsystems accurately and efficiently. To achieve maximal efficiency, it is convenient to adopt a hybrid scheme, where the QM electron density is used explicitly in the evaluation of short-range QM/MM electrostatic interactions, while a multipolar representation for the QM electron density is employed to account for the long-range QM/MM electrostatic interactions. In order to avoid energy discontinuity at the cutoffs, which separate the short- and long-range QM/MM electrostatic interactions, a switching function should be utilized to ensure a smooth potential energy surface. In this study, we benchmarked the accuracy of such hybrid embedding schemes for QM/MM electrostatic interactions using different multipolar representations, switching functions and cutoff distances. For test systems (neutral and anionic oxyluciferin in MM (aqueous and enzyme) environments), the best accuracy was acquired with a combination of QM electrostatic potential (ESP) charges and dipoles and two switching functions (long-range electrostatic corrections (LREC) and Switch) in the treatment of long-range QM/MM electrostatics. It allowed us to apply a 10Å distance cutoff and still obtain QM/MM electrostatics/polarization energies within 0.1 kcal/mol and time-dependent density functional theory (TDDFT)/MM vertical excitation energies within 10−3 eV from theoretical reference values.
Open Access
Ring-like N-fold Models of Aβ42 fibrils
(2017-07-26) Xi, Wenhui; Hansmann, Ulrich H. E.
When assembling as fibrils Aβ40 peptides can only assume U-shaped conformations while Aβ42 can also arrange as S-shaped three-stranded chains. We show that this allows Aβ42 peptides to assemble pore-like structures that may explain their higher toxicity. For this purpose, we develop a scalable model of ring-like assemblies of S-shaped Aβ1–42 chains and study the stability and structural properties of these assemblies through atomistic molecular dynamics simulations. We find that the proposed arrangements are in size and symmetry compatible with experimentally observed Aβ assemblies. We further show that the interior pore in our models allows for water leakage as a possible mechanism of cell toxicity of Aβ42 amyloids.
Open Access
Top-down Mass Spectrometry Analysis of Human Serum Autoantibody Antigen-Binding Fragments
(2019-02-20) Wang, Zhe; Liu, Xiaowen; Muther, Jennifer; James, Judith A.; Smith, Kenneth; Wu, Si
Detecting autoimmune diseases at an early stage is crucial for effective treatment and disease management to slow disease progression and prevent irreversible organ damage. In many autoimmune diseases, disease-specific autoantibodies are produced by B cells in response to soluble autoantigens due to defects in B cell tolerance mechanisms. Autoantibodies accrue early in disease development, and several are so disease-specific they serve as classification criteria. In this study, we established a high-throughput, sensitive, intact serum autoantibody analysis platform based on the optimization of a one dimensional ultra-high-pressure liquid chromatography top-down mass spectrometry platform (1D UPLC-TDMS). This approach has been successfully applied to a 12 standard monoclonal antibody antigen-binding fragment (Fab) mixture, demonstrating the feasibility to separate and sequence intact antibodies with high sequence coverage and high sensitivity. We then applied the optimized platform to characterize total serum antibody Fabs in a systemic lupus erythematosus (SLE) patient sample and compared it to healthy control samples. From this analysis, we show that the SLE sample has many dominant antibody Fab-related mass features unlike the healthy controls. To our knowledge, this is the first top-down demonstration of serum autoantibody pool analysis. Our proposed approach holds great promise for discovering novel serum autoantibody biomarkers that are of interest for diagnosis, prognosis, and tolerance induction, as well as improving our understanding of pathogenic autoimmune processes.
Open Access
A YoeB toxin cleaves both RNA and DNA
(2021-02-11) McGillick, Julia; Ames, Jessica R.; Murphy, Tamiko; Bourne, Christina R.
Type II toxin-antitoxin systems contain a toxin protein, which mediates diverse interactions within the bacterial cell when it is not bound by its cognate antitoxin protein. These toxins provide a rich source of evolutionarily-conserved tertiary folds that mediate diverse catalytic reactions. These properties make toxins of interest in biotechnology applications, and studies of the catalytic mechanisms continue to provide surprises. In the current work, our studies on a YoeB family toxin from Agrobacterium tumefaciens have revealed a conserved ribosome-independent non-specific nuclease activity. We have quantified the RNA and DNA cleavage activity, revealing they have essentially equivalent dose-dependence while differing in requirements for divalent cations and pH sensitivity. The DNA cleavage activity is as a nickase for any topology of double-stranded DNA, as well as cleaving single-stranded DNA. AtYoeB is able to bind to double-stranded DNA with mid-micromolar affinity. Comparison of the ribosome-dependent and -independent reactions demonstrates an approximate tenfold efficiency imparted by the ribosome. This demonstrates YoeB toxins can act as non-specific nucleases, cleaving both RNA and DNA, in the absence of being bound within the ribosome.