Browsing by Author "Bothner, Brian"

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Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking
(2020-06) Lins-Austin, Bridget; Patel, Saajan; Mietzsch, Mario; Brooke, Dewey; Bennett, Antonette; Venkatakrishnan, Balasubramanian; Van Vliet, Kim; Smith, Adam N.; Long, Joanna R.; McKenna, Robert; Potter, Mark; Byrne, Barry; Boye, Sanford L.; Bothner, Brian; Heilbronn, Regine; Agbandje-McKenna, Mavis
Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.
Aerobic bacterial methane synthesis
(Proceedings of the National Academy of Sciences, 2021-06) Wang, Qian; Alowaifeer, Abdullah; Kerner, Patricia; Balasubramanian, Narayanaganesh; Patterson, Angela; Christian, William; Tarver, Angela; Dore, John E.; Hatzenpichler, Roland; Bothner, Brian; McDermott, Timothy R.
Reports of biogenic methane (CH4) synthesis associated with a range of organisms have steadily accumulated in the literature. This has not happened without controversy and in most cases the process is poorly understood at the gene and enzyme levels. In marine and freshwater environments, CH4 supersaturation of oxic surface waters has been termed the “methane paradox” because biological CH4 synthesis is viewed to be a strictly anaerobic process carried out by O2-sensitive methanogens. Interest in this phenomenon has surged within the past decade because of the importance of understanding sources and sinks of this potent greenhouse gas. In our work on Yellowstone Lake in Yellowstone National Park, we demonstrate microbiological conversion of methylamine to CH4 and isolate and characterize an Acidovorax sp. capable of this activity. Furthermore, we identify and clone a gene critical to this process (encodes pyridoxylamine phosphate-dependent aspartate aminotransferase) and demonstrate that this property can be transferred to Escherichia coli with this gene and will occur as a purified enzyme. This previously unrecognized process sheds light on environmental cycling of CH4, suggesting that O2-insensitive, ecologically relevant aerobic CH4 synthesis is likely of widespread distribution in the environment and should be considered in CH4 modeling efforts.
Aerobic methane synthesis and dynamics in a river water environment
(Wiley, 2023-06) Alowaifeer, Abdullah M.; Wang, Qian; Bothner, Brian; Sibert, Ryan J.; Joye, Samantha B.; McDermott, Timothy R.
Reports of aerobic biogenic methane (CH4) have generated new views about CH4 sources in nature. We examine this phenomenon in the free-flowing Yellowstone river wherein CH4 concentrations were tracked as a function of environmental conditions, phototrophic microorganisms (using chlorophyll a, Chl a, as proxy), as well as targeted methylated amines known to be associated with this process. CH4 was positively correlated with temperature and Chl a, although diurnal measurements showed CH4 concentrations were greatest during the night and lowest during maximal solar irradiation. CH4 efflux from the river surface was greater in quiescent edge waters (71–94 μmol m−2 d) than from open flowing current (~ 57 μmol m−2 d). Attempts to increase flux by disturbing the benthic environment in the quiescent water directly below (~ 1.0 m deep) or at varying distances (0–5 m) upstream of the flux chamber failed to increase surface flux. Glycine betaine (GB), dimethylamine and methylamine (MMA) were observed throughout the summer-long study, increasing during a period coinciding with a marked decline in Chl a, suggesting a lytic event led to their release; however, this did not correspond to increased CH4 concentrations. Spiking river water with GB or MMA yielded significantly greater CH4 than nonspiked controls, illustrating the metabolic potential of the river microbiome. In summary, this study provides evidence that: (1) phototrophic microorganisms are involved in CH4 synthesis in a river environment; (2) the river microbiome possesses the metabolic potential to convert methylated amines to CH4; and (3) river CH4 concentrations are dynamic diurnally as well as during the summer active months.
Altered stoichiometry Escherichia coli Cascade complexes with shortened CRISPR RNA spacers are capable of interference and primed adaptation
(2016-10) Kuznedelov, Konstantin; Mekler, Vladimir; Lemak, Sofia; Tokmina-Lukaszewska, Monika; Datsenko, Kirill A; Jain, Ishita; Savitskaya, Ekaterina; Mallon, John; Shmakov, Sergey; Bothner, Brian; Bailey, Scott; Yakunin, Alexander F; Severinov, Konstantin
The Escherichia coli type I-E CRISPR-Cas system Cascade effector is a multisubunit complex that binds CRISPR RNA (crRNA). Through its 32-nucleotide spacer sequence, Cascade-bound crRNA recognizes protospacers in foreign DNA, causing its destruction during CRISPR interference or acquisition of additional spacers in CRISPR array during primed CRISPR adaptation. Within Cascade, the crRNA spacer interacts with a hexamer of Cas7 subunits. We show that crRNAs with a spacer length reduced to 14 nucleotides cause primed adaptation, while crRNAs with spacer lengths of more than 20 nucleotides cause both primed adaptation and target interference in vivo Shortened crRNAs assemble into altered-stoichiometry Cascade effector complexes containing less than the normal amount of Cas7 subunits. The results show that Cascade assembly is driven by crRNA and suggest that multisubunit type I CRISPR effectors may have evolved from much simpler ancestral complexes.
Analysis of phospholipase activity in adeno-associated virus particles by liquid-chromatography/mass-spectrometry
(2013-03) Brooke, Dewey; Bothner, Brian; Agbandje-McKenna, Mavis
Adeno-associated virus (AAV) belongs to the Parvovridae, a family of small, non-enveloped isosahedral viruses. The viral capsid has T=1 symmetry and is composed of 60 subunits, made up from three proteins (VP1, VP2, VP3) in a ratio of 1:1:10. The minor proteins are the same as VP3 in their C-termini region, but they have additional domains on their N-termini that play essential roles in cellular entry and trafficking. Structural studies of AAV have shown that the N-termini of VP1 and VP2 are initially internalized in the capsid and become externalized, most likely during endocytosis. Based on sequence and structural similarity, VP1 contains a phospholipase A2 domain (PLA2) which, when mutated, dramatically reduces infectivity. Currently, little is known about the mechanism of VP1 externalization or the role of the lipase in escape of the virus particle from the endosome. Also, due to low sequence similarity, there is even concern over whether this is a true PLA2 type domain. To address these questions, we have developed a liquid-chromatography mass-spectrometry based assay for lipase activity. To date, we have tested factors such as receptor binding, heat, and pH on the externalization of the PLA2 and are addressing the question of substrate specificity.
Anti-CRISPR proteins function through thermodynamic tuning and allosteric regulation of CRISPR RNA-guided surveillance complex
(Oxford University Press, 2022-10) Patterson, Angela; White, Aidan; Waymire, Elizabeth; Fleck, Sophie; Golden, Sarah; Wilkinson, Royce A.; Wiedenheft, Blake; Bothner, Brian
CRISPR RNA-guided detection and degradation of foreign DNA is a dynamic process. Viruses can interfere with this cellular defense by expressing small proteins called anti-CRISPRs. While structural models of anti-CRISPRs bound to their target complex provide static snapshots that inform mechanism, the dynamics and thermodynamics of these interactions are often overlooked. Here, we use hydrogen deuterium exchange-mass spectrometry (HDX-MS) and differential scanning fluorimetry (DSF) experiments to determine how anti-CRISPR binding impacts the conformational landscape of the type IF CRISPR RNA guided surveillance complex (Csy) upon binding of two different anti-CRISPR proteins (AcrIF9 and AcrIF2). The results demonstrate that AcrIF2 binding relies on enthalpic stabilization, whereas AcrIF9 uses an entropy driven reaction to bind the CRISPR RNA-guided surveillance complex. Collectively, this work reveals the thermodynamic basis and mechanistic versatility of anti-CRISPR-mediated immune suppression. More broadly, this work presents a striking example of how allosteric effectors are employed to regulate nucleoprotein complexes.
Arsenate-Induced Changes in Bacterial Metabolite and Lipid Pools during Phosphate Stress
(American Society for Microbiology, 2021-02) Zhuang, Weiping; Balasubramanian, Narayanaganesh; Wang, Lu; Wang, Qian; McDermott, Timothy R.; Copie, Valerie; Wang, Gejiao; Bothner, Brian
Arsenic is widespread in the environment and is one of the most ubiquitous environmental pollutants. Parodoxically, the growth of certain bacteria is enhanced by arsenic when phosphate is limited.
Arsenic Exposure Causes Global Changes in the Metalloproteome of Escherichia coli
(MDPI AG, 2023-02) Larson, James; Tokmina-Lukaszewska, Monika; Fausset, Hunter; Spurzem, Scott; Cox, Savannah; Cooper, Gwendolyn; Copié, Valérie; Bothner, Brian
Arsenic is a toxic metalloid with differential biological effects, depending on speciation and concentration. Trivalent arsenic (arsenite, AsIII) is more toxic at lower concentrations than the pentavalent form (arsenate, AsV). In E. coli, the proteins encoded by the arsRBC operon are the major arsenic detoxification mechanism. Our previous transcriptional analyses indicate broad changes in metal uptake and regulation upon arsenic exposure. Currently, it is not known how arsenic exposure impacts the cellular distribution of other metals. This study examines the metalloproteome of E. coli strains with and without the arsRBC operon in response to sublethal doses of AsIII and AsV. Size exclusion chromatography coupled with inductively coupled plasma mass spectrometry (SEC-ICPMS) was used to investigate the distribution of five metals (56Fe, 24Mg, 66Zn, 75As, and 63Cu) in proteins and protein complexes under native conditions. Parallel analysis by SEC-UV-Vis spectroscopy monitored the presence of protein cofactors. Together, these data reveal global changes in the metalloproteome, proteome, protein cofactors, and soluble intracellular metal pools in response to arsenic stress in E. coli. This work brings to light one outcome of metal exposure and suggests that metal toxicity on the cellular level arises from direct and indirect effects.
An Aurora B-RPA signaling axis secures chromosome segregation fidelity
(Springer Science and Business Media LLC, 2023-05) Roshan, Poonam; Kuppa, Sahiti; Mattice, Jenna R.; Kaushik, Vikas; Chadda, Rahul; Pokhrel, Nilisha; Tumala, Brunda R.; Biswas, Aparna; Bothner, Brian; Antony, Edwin; Origanti, Sofia
Errors in chromosome segregation underlie genomic instability associated with cancers. Resolution of replication and recombination intermediates and protection of vulnerable single-stranded DNA (ssDNA) intermediates during mitotic progression requires the ssDNA binding protein Replication Protein A (RPA). However, the mechanisms that regulate RPA specifically during unperturbed mitotic progression are poorly resolved. RPA is a heterotrimer composed of RPA70, RPA32 and RPA14 subunits and is predominantly regulated through hyperphosphorylation of RPA32 in response to DNA damage. Here, we have uncovered a mitosis-specific regulation of RPA by Aurora B kinase. Aurora B phosphorylates Ser-384 in the DNA binding domain B of the large RPA70 subunit and highlights a mode of regulation distinct from RPA32. Disruption of Ser-384 phosphorylation in RPA70 leads to defects in chromosome segregation with loss of viability and a feedback modulation of Aurora B activity. Phosphorylation at Ser-384 remodels the protein interaction domains of RPA. Furthermore, phosphorylation impairs RPA binding to DSS1 that likely suppresses homologous recombination during mitosis by preventing recruitment of DSS1-BRCA2 to exposed ssDNA. We showcase a critical Aurora B-RPA signaling axis in mitosis that is essential for maintaining genomic integrity.
Biophysical characterization of SARAH domain–mediated multimerization of Hippo pathway complexes in Drosophila
(2020-05) Cairns, Leah; Patterson, Angela; Weingartner, Kyler A.; Koehler, T. J.; DeAngelis, Daniel R.; Tripp, Katherine W.; Bothner, Brian; Kavran, Jennifer M.
Hippo pathway signaling limits cell growth and proliferation and maintains the stem-cell niche. These cellular events result from the coordinated activity of a core kinase cassette that is regulated, in part, by interactions involving Hippo, Salvador, and dRassF. These interactions are mediated by a conserved coiled-coil domain, termed SARAH, in each of these proteins. SARAH domain–mediated homodimerization of Hippo kinase leads to autophosphorylation and activation. Paradoxically, SARAH domain–mediated heterodimerization between Hippo and Salvador enhances Hippo kinase activity in cells, whereas complex formation with dRassF inhibits it. To better understand the mechanism by which each complex distinctly modulates Hippo kinase and pathway activity, here we biophysically characterized the entire suite of SARAH domain–mediated complexes. We purified the three SARAH domains from Drosophila melanogaster and performed an unbiased pulldown assay to identify all possible interactions, revealing that isolated SARAH domains are sufficient to recapitulate the cellular assemblies and that Hippo is a universal binding partner. Additionally, we found that the Salvador SARAH domain homodimerizes and demonstrate that this interaction is conserved in Salvador's mammalian homolog. Using native MS, we show that each of these complexes is dimeric in solution. We also measured the stability of each SARAH domain complex, finding that despite similarities at both the sequence and structural levels, SARAH domain complexes differ in stability. The identity, stoichiometry, and stability of these interactions characterized here comprehensively reveal the nature of SARAH domain–mediated complex formation and provide mechanistic insights into how SARAH domain–mediated interactions influence Hippo pathway activity.
The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD
(2018-12) Schut, Gerrit J.; Mohamed-Raseek, Nishya; Tokmina-Lukaszewska, Monika; Mulder, David W.; Nguyen, Diep M. N.; Lipscomb, Gina L.; Hoben, John P.; Patterson, Angela; Lubner, Carolyn E.; King, Paul W.; Peters, John W.; Bothner, Brian; Miller, Anne-Frances; Adams, Michael W. W.
Electron bifurcation plays a key role in anaerobic energy metabolism but it is a relatively new discovery and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using non-denaturing MS, cross-linking and homology modeling in which EtfA, B, and C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically-unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and in the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETF\'s and can be applied to the large bifurcating ETF family.
Changes in the stability and biomechanics of P22 bacteriophage capsid during maturation
(2018-06) Kant, Ravi; Llauro, Aida; Rayaprolu, Vamseedhar; Qazi, Shefah; de Pablo, Pedro J.; Douglas, Trevor; Bothner, Brian
The capsid of P22 bacteriophage undergoes a series of structural transitions during maturation that guide it from spherical to icosahedral morphology. The transitions include the release of scaffold proteins and capsid expansion. Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage. To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry. By integrating these results from single particle and population measurements, an energy landscape of P22 maturation from procapsid through expanded shell to wiffle ball emerged, highlighting the role of metastable structures and the thermodynamics guiding maturation. The propagation of weak quaternary interactions across symmetric elements of the capsid is a key component for stability in P22. A surprising finding is that the progression to wiffle ball, which lacks pentamers, shows that chemical and thermal stability can be uncoupled from mechanical rigidity, elegantly demonstrating the complexity inherent in capsid protein interactions and the emergent properties that can arise from icosahedral symmetry. On a broader scale, this work demonstrates the power of applying orthogonal biophysical techniques to elucidate assembly mechanisms for supramolecular complexes and provides a framework within which other viral systems can be compared.
Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis
(2019-08) Carlson, Alyssa K.; Rawle, Rachel A.; Wallace, Cameron W.; Brooks, Ellen G.; Adams, Erik; Greenwood, Mark C.; Olmer, Merissa; Lotz, Martin K.; Bothner, Brian; June, Ronald K.
"Objective Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. Design: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. Results: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. Conclusion: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.
Constitutive redox and phosphoproteome changes in multiple herbicide resistant Avena fatua L. are similar to those of systemic acquired resistance and systemic acquired acclimation
(2018-01) Burns, Erin E.; Keith, Barbara K.; Mohammed, Refai Y.; Bothner, Brian; Dyer, William E.
Plants are routinely confronted with numerous biotic and abiotic stressors, and in response have evolved highly effective strategies of systemic acquired resistance (SAR) and systemic acquired acclimation (SAA), respectively. A much more evolutionarily recent abiotic stress is the application of herbicides to control weedy plants, and their intensive use has selected for resistant weed populations that cause substantial crop yield losses and increase production costs. Non-target site resistance (NTSR) to herbicides is rapidly increasing worldwide and is associated with alterations in generalized stress defense networks. This work investigated protein post-translational modifications associated with NTSR in multiple herbicide resistant (MHR) Avena fatua, and their commonalities with those of SAR and SAA. We used proteomic, biochemical, and immunological approaches to compare constitutive protein profiles in MHR and herbicide susceptible (HS) A. fatua populations. Phosphoproteome and redox proteome surveys showed that post-translational modifications of proteins with functions in core cellular processes were reduced in MHR plants, while those involved in xenobiotic and stress response, reactive oxygen species detoxification and redox maintenance, heat shock response, and intracellular signaling were elevated in MHR as compared to HS plants. More specifically, MHR plants contained constitutively elevated levels of three protein kinases including the lectin S-receptor-like serine/threonine-protein kinase LecRK2, a well-characterized component of SAR. Analyses of superoxide dismutase enzyme activity and protein levels did not reveal constitutive differences between MHR and HS plants. The overall results support the idea that herbicide stress is perceived similarly to other abiotic stresses, and that A. fatua NTSR shares analogous features with SAR and SAA. We speculate that MHR A. fatua's previous exposure to sublethal herbicide doses, as well as earlier evolution under a diversity of abiotic and biotic stressors, has led to a heightened state of stress preparedness that includes NTSR to a number of unrelated herbicides.
Core Protein-Directed Antivirals and Importin β Can Synergistically Disrupt Hepatitis B Virus Capsids
(American Society for Microbiology, 2022-01) Kim, Christine; Barnes, Lauren F.; Schlicksup, Christopher J.; Patterson, Angela J.; Bothner, Brian; Jarrold, Martin F.; Wang, Che-Yen Joseph; Zlotnick, Adam
The HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to “flip” to the capsid exterior.
Curating viscoelastic properties of icosahedral viruses, virus-based nanomaterials, and protein cages
(2018-06) Kant, Ravi; Rayaprolu, Vamseedhar; McDonald, Kaitlyn; Bothner, Brian
The beauty, symmetry, and functionality of icosahedral virus capsids has attracted the attention of biologists, physicists, and mathematicians ever since they were first observed. Viruses and protein cages assemble into functional architectures in a range of sizes, shapes, and symmetries. To fulfill their biological roles, these structures must self-assemble, resist stress, and are often dynamic. The increasing use of icosahedral capsids and cages in materials science has driven the need to quantify them in terms of structural properties such as rigidity, stiffness, and viscoelasticity. In this study, we employed Quartz Crystal Microbalance with Dissipation technology (QCM-D) to characterize and compare the mechanical rigidity of different protein cages and viruses. We attempted to unveil the relationships between rigidity, radius, shell thickness, and triangulation number. We show that the rigidity and triangulation numbers are inversely related to each other and the comparison of rigidity and radius also follows the same trend. Our results suggest that subunit orientation, protein–protein interactions, and protein–nucleic acid interactions are important for the resistance to deformation of these complexes, however, the relationships are complex and need to be explored further. The QCM-D based viscoelastic measurements presented here help us elucidate these relationships and show the future prospect of this technique in the field of physical virology and nano-biotechnology.
Decrease in pH destabilizes individual vault nanocages by weakening the inter-protein lateral interaction
(2016-10) Llauro, Aida; Guerra, Pablo; Kant, Ravi; Bothner, Brian; Verdaguer, Nuria; de Pablo, Pedro J
Vault particles are naturally occurring proteinaceous cages with promising application as molecular containers. The use of vaults as functional transporters requires a profound understanding of their structural stability to guarantee the protection and controlled payload delivery. Previous results performed with bulk techniques or at non-physiological conditions have suggested pH as a parameter to control vault dynamics. Here we use Atomic Force Microscopy (AFM) to monitor the structural evolution of individual vault particles while changing the pH in real time. Our experiments show that decreasing the pH of the solution destabilize the barrel region, the central part of vault particles, and leads to the aggregation of the cages. Additional analyses using Quartz-Crystal Microbalance (QCM) and Differential Scanning Fluorimetry (DSF) are consistent with our single molecule AFM experiments. The observed topographical defects suggest that low pH weakens the bonds between adjacent proteins. We hypothesize that the observed effects are related to the strong polar character of the protein-protein lateral interactions. Overall, our study unveils the mechanism for the influence of a biologically relevant range of pHs on the stability and dynamics of vault particles.
Detection of Pseudomonas aeruginosa biomarkers from thermally injured mice in situ using imaging mass spectrometry
(2017-12) Hamerly, Timothy; Everett, Jake A.; Paris, Nina; Fisher, Steve T.; Karunamurthy, Arivarasan; James, Garth A.; Rumbaugh, Kendra P.; Rhoads, Daniel D.; Bothner, Brian
Monitoring patients with burn wounds for infection is standard practice because failure to rapidly and specifically identify a pathogen can result in poor clinical outcomes, including death. Therefore, a method that facilitates detection and identification of pathogens in situ within minutes of biopsy would be a significant benefit to clinicians. Mass spectrometry is rapidly becoming a standard tool in clinical settings, capable of identifying specific pathogens from complex samples. Imaging mass spectrometry (IMS) expands the information content by enabling spatial resolution of biomarkers in tissue samples as in histology, without the need for specific stains/antibodies. Herein, a murine model of thermal injury was used to study infection of burn tissue by Pseudomonas aeruginosa. This is the first use of IMS to detect P. aeruginosa infection in situ from thermally injured tissue. Multiple molecular features could be spatially resolved to infected or uninfected tissue. This demonstrates the potential use of IMS in a clinical setting to aid doctors in identifying both presence and species of pathogens in tissue.
Differences in amino acid catabolism by gut microbes with/without prebiotics inclusion in GDDY-based diet affect feed utilization in rainbow trout
(2017-09) Betiku, Omolola C.; Yeoman, Carl J.; Gaylord, T. Gibson; Duff, Glenn C.; Hamerly, Timothy; Bothner, Brian; Block, Stephanie S.; Sealey, Wendy M.
There is the need to enhance feed efficiency and growth of rainbow trout to reduce production costs of cultured fish. This study conducted a 3 × 4 factorial experiment with three graded levels of grain distiller dried yeast (GDDY) protein (0%, 50%, 75%) as replacement for fishmeal and four different prebiotics inclusion levels (0% (control), 0.4%, 1% mannooligosaccharides (MOS), and 1% GroBiotic A). The feeding trial was conducted for 12 weeks during which fish were fed daily to apparent satiation. Growth of rainbow trout was not affected by replacement of fishmeal with GDDY, but feed conversion ratio (P < 0.0001) was greater at the highest level of GDDY inclusion. Increasing GDDY inclusion significantly increased feed intake (P < 0.00015), which resulted in poor feed utilization. Acetic (P = 0.1994), propionic (P = 0.8037), butyric (P = 0.6268), valeric (P = 0.5877), and isovaleric (P = 0.5919) acids profiles did not differ by diet nor with inclusion of MOS or GroBiotic A. Whole shotgun metagenomic analyses of the gastrointestinal tract (GIT) microbiota revealed enrichment in the fungal phyla Ascomycota and Basidiomycota and the bacterial phylum Actinobacteria in the GDDY-fed fish compared to those fed the control fishmeal-based diet, which may be reflective of the species endogenous in GDDY. Microbial genes involved in branched-chain amino acid metabolism (glutamate, glutamine, aspartate) (P = 0.028) and glutamate dehydrogenase clusters (P = 0.0192), were also elevated in the fish fed the 75% GDDY-based diet. The results from this study indicate the potential for microbially-mediated catabolism of the non-essential amino acids, and suggest this activity may significantly influence efficient utilization of dietary nitrogen in the yeast-based protein diet.
Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting
(MDPI AG, 2022-11) Kempa, Jacob; O’Shea-Stone, Galen; Moss, Corinne E.; Peters, Tami; Marcotte, Tamera K.; Tripet, Brian; Eilers, Brian; Bothner, Brian; Copié, Valérie; Pincus, Seth H.
Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.