Scholarly Works, Biochemistry

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  • Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase
    Johnson, Sydney B.; Li, Hao; Valentino, Hannah; Sobrado, Pablo (American Chemical Society, 2024-05-23)
    OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a “cautious” flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme−substrate and enzyme− intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.
  • The Essential Oils Compounds of Lippia multiflora Moldenke and Cymbopogon schoenanthus (L.) Spreng Repel and Affect the Survival of the Maize Pest Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae
    Wangrawa, Dimitri Wendgida; Waongo, Antoine; Traore, Fousséni; Sanou, Drissa; Lahondere, Chloé; Sané, Abdoul Razac; Borovsky, Dov; Ouédraogo, Sylvain Nafita; Sanon, Antoine (Hindawi, 2024-04-01)
    Plant-derived insecticides, such as essential oils, can be an effective alternative to replace synthetic chemical insecticides against Spodoptera frugiperda, which becomes increasingly resistant to synthetic products. This study aims to evaluate essential oils (EOs) effects on larval growth and development following feeding inhibition, growth regulation, and repellency of EOs of Lippia multiflora (Verbenaceae), Cymbopogon schoenanthus (Poaceae), and their combination. Topical application of EOs was used on S. frugiperda larvae for larvicidal effect or treating filter paper with the EOs to find repellency. The effect of EOs on food intake and larval growth was also evaluated. Several types of compounds have been identified in the EOs, mainly monoterpenes with the appearance of new compounds in the 1 : 1 combination. Bioassay results show that individuals and combinations of EOs significantly influenced S. frugiperda larval development. L. multiflora caused 100% mortality of L2 larvae within 24 hours at 3%. The Lm + Cs (1 : 1) EOs combination was the most effective with LC50 and LC90 of 1.02% and 1.92%, respectively. Lm + Cs (1/4 : 3/4) EOs caused the highest inhibition of food consumption, 0.0160 g consumed after food was treated with 2.2% concentration compared to food consumption of 0.0602 g for the control group at 24 hours. Lower food consumption caused the inhibition of larval growth and weight loss of 0.0005 g/day at the 2.2% EOs concentration. The highest repellency effect of the EOs was found in EOs of L. multiflora, exhibiting a repulsion of 83.33% of the larvae after 3 hours of exposure. This diversity in the biological actions of the EOs tested on S. frugiperda represents valuable options for contributing to integrated pest management and an alternative to synthetic chemical insecticides.
  • Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation
    Holub, Meaghan N.; Wahhab, Amanda; Rouse, Joseph R.; Danner, Rebecca; Hackner, Lauren G.; Duris, Christine B.; McClune, Mecaila E.; Dressler, Jules M.; Strle, Klemen; Jutras, Brandon L.; Edelstein, Adam I.; Lochhead, Robert B. (2024-03-27)
    Objectives: Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. Methods: Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. Results: A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. Conclusion: Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.
  • Sinorhizobium meliloti Chemoreceptor McpV Senses Short-Chain Carboxylates via Direct Binding
    Compton, K. Karl; Hildreth, Sherry B.; Helm, Richard F.; Scharf, Birgit E. (2018-12)
    Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels and chemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR. Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPR were in the micromolar range. An McpVPR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction.
  • More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1
    Gonzalez, Floricel; Helm, Richard F.; Broadway, Katherine M.; Scharf, Birgit E. (2018-10)
    Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.
  • The chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus uncovers patterns of genome evolution in mosquitoes
    Ryazansky, Sergei S.; Chen, Chujia; Potters, Mark; Naumenko, Anastasia N.; Lukyanchikova, Varvara; Masri, Reem A.; Brusentsov, Ilya I.; Karagodin, Dmitriy A.; Yurchenko, Andrey A.; dos Anjos, Vitor L.; Haba, Yuki; Rose, Noah H.; Hoffman, Jinna; Guo, Rong; Menna, Theresa; Kelley, Melissa; Ferrill, Emily; Schultz, Karen E.; Qi, Yumin; Sharma, Atashi; Deschamps, Stéphane; Llaca, Victor; Mao, Chunhong; Murphy, Terence D.; Baricheva, Elina M.; Emrich, Scott; Fritz, Megan L.; Benoit, Joshua B.; Sharakhov, Igor V.; McBride, Carolyn S.; Tu, Zhijian; Sharakhova, Maria V. (2024-01-25)
    Background: Understanding genome organization and evolution is important for species involved in transmission of human diseases, such as mosquitoes. Anophelinae and Culicinae subfamilies of mosquitoes show striking differences in genome sizes, sex chromosome arrangements, behavior, and ability to transmit pathogens. However, the genomic basis of these differences is not fully understood. Methods: In this study, we used a combination of advanced genome technologies such as Oxford Nanopore Technology sequencing, Hi-C scaffolding, Bionano, and cytogenetic mapping to develop an improved chromosome-scale genome assembly for the West Nile vector Culex quinquefasciatus. Results: We then used this assembly to annotate odorant receptors, odorant binding proteins, and transposable elements. A genomic region containing male-specific sequences on chromosome 1 and a polymorphic inversion on chromosome 3 were identified in the Cx. quinquefasciatus genome. In addition, the genome of Cx. quinquefasciatus was compared with the genomes of other mosquitoes such as malaria vectors An. coluzzi and An. albimanus, and the vector of arboviruses Ae. aegypti. Our work confirms significant expansion of the two chemosensory gene families in Cx. quinquefasciatus, as well as a significant increase and relocation of the transposable elements in both Cx. quinquefasciatus and Ae. aegypti relative to the Anophelines. Phylogenetic analysis clarifies the divergence time between the mosquito species. Our study provides new insights into chromosomal evolution in mosquitoes and finds that the X chromosome of Anophelinae and the sex-determining chromosome 1 of Culicinae have a significantly higher rate of evolution than autosomes. Conclusion: The improved Cx. quinquefasciatus genome assembly uncovered new details of mosquito genome evolution and has the potential to speed up the development of novel vector control strategies.
  • Differences in Conformational Sampling and Intrinsic Electric Fields Drive Ion Binding in Telomeric and TERRA G-Quadruplexes
    Poleto, Marcelo D.; Lemkul, Justin A. (American Chemical Society, 2023-10-17)
    The formation of G-quadruplexes (GQs) occurs in guanine-rich sequences of DNA and RNA, producing highly stable and structurally diverse noncanonical nucleic acid structures. GQs play crucial roles in regulating transcription, translation, and replication and maintaining the genome, among others; thus, changes to their structures can lead to diseases such as cancer. Previous studies using polarizable molecular dynamics simulations have shown differences in ion binding properties between telomeric and telomeric repeat-containing RNA GQs despite architectural similarities. Here, we used volume-based metadynamics and repulsive potential simulations in conjunction with polarizable force fields to quantify the impact of ion binding on the GQ dynamics and ion binding free energies. Furthermore, we describe how GQs exert electric fields on their surroundings to link dynamics with variations in the electronic structure. Our findings provide new insights into the energetic, physical, and conformational properties of GQs and expose subtle but important differences between DNA and RNA GQs with the same fold.
  • An in silico approach to determine inter-subunit affinities in human septin complexes
    Grupp, Benjamin; Lemkul, Justin A.; Gronemeyer, Thomas (Wiley, 2023-03-09)
    The septins are a conserved family of filament-forming guanine nucleotide binding proteins, often named the fourth component of the cytoskeleton. Correctly assembled septin structures are required for essential intracellular processes such as cytokinesis, vesicular transport, polarity establishment, and cellular adhesion. Structurally, septins belong to the P-Loop NTPases but they do not mediate signals to effectors through GTP binding and hydrolysis. GTP binding and hydrolysis are believed to contribute to septin complex integrity, but biochemical approaches addressing this topic are hampered by the stability of septin complexes after recombinant expression and the lack of nucleotide-depleted complexes. To overcome this limitation, we used a molecular dynamics-based approach to determine inter-subunit binding free energies in available human septin dimer structures and in their apo forms, which we generated in silico. The nucleotide in the GTPase active subunits SEPT2 and SEPT7, but not in SEPT6, was identified as a stabilizing element in the G interface. Removal of GDP from SEPT2 and SEPT7 results in flipping of a conserved Arg residue and disruption of an extensive hydrogen bond network in the septin unique element, concomitant with a decreased inter-subunit affinity. Based on these findings we propose a singular “lock-hydrolysis” mechanism stabilizing human septin filaments.
  • Evolving understanding of rumen methanogen ecophysiology
    Khairunisa, Bela Haifa; Heryakusuma, Christian; Ike, Kelechi; Mukhopadhyay, Biswarup; Susanti, Dwi (Frontiers, 2023-11-06)
    Production of methane by methanogenic archaea, or methanogens, in the rumen of ruminants is a thermodynamic necessity for microbial conversion of feed to volatile fatty acids, which are essential nutrients for the animals. On the other hand, methane is a greenhouse gas and its production causes energy loss for the animal. Accordingly, there are ongoing efforts toward developing effective strategies for mitigating methane emissions from ruminant livestock that require a detailed understanding of the diversity and ecophysiology of rumen methanogens. Rumen methanogens evolved from free-living autotrophic ancestors through genome streamlining involving gene loss and acquisition. The process yielded an oligotrophic lifestyle, and metabolically efficient and ecologically adapted descendants. This specialization poses serious challenges to the efforts of obtaining axenic cultures of rumen methanogens, and consequently, the information on their physiological properties remains in most part inferred from those of their non-rumen representatives. This review presents the current knowledge of rumen methanogens and their metabolic contributions to enteric methane production. It also identifies the respective critical gaps that need to be filled for aiding the efforts to mitigate methane emission from livestock operations and at the same time increasing the productivity in this critical agriculture sector.
  • Increased Fusobacterium tumoural abundance affects immunogenicity in mucinous colorectal cancer and may be associated with improved clinical outcome
    Duggan, William P.; Salvucci, Manuela; Kisakol, Batuhan; Lindner, Andreas U.; Reynolds, Ian S.; Dussmann, Heiko; Fay, Joanna; O'Grady, Tony; Longley, Daniel B.; Ginty, Fiona; McDonough, Elizabeth; Slade, Daniel J.; Burke, John P.; Prehn, Jochen H. M. (Springer, 2023-07)
    Abstract: There is currently an urgent need to identify factors predictive of immunogenicity in colorectal cancer (CRC). Mucinous CRC is a distinct histological subtype of CRC, associated with a poor response to chemotherapy. Recent evidence suggests the commensal facultative anaerobe Fusobacterium may be especially prevalent in mucinous CRC. The objectives of this study were to assess the association of Fusobacterium abundance with immune cell composition and prognosis in mucinous CRC. Our study included two independent colorectal cancer patient cohorts, The Cancer Genome Atlas (TCGA) cohort, and a cohort of rectal cancers from the Beaumont RCSI Cancer Centre (BRCC). Multiplexed immunofluorescence staining of a tumour microarray (TMA) from the BRCC cohort was undertaken using Cell DIVE technology. Our cohorts included 87 cases (13.3%) of mucinous and 565 cases (86.7%) of non-mucinous CRC. Mucinous CRC in the TCGA dataset was associated with an increased proportion of CD8 + lymphocytes (p = 0.018), regulatory T-cells (p = 0.001) and M2 macrophages (p = 0.001). In the BRCC cohort, mucinous RC was associated with enhanced CD8 + lymphocyte (p = 0.022), regulatory T-cell (p = 0.047), and B-cell (p = 0.025) counts. High Fusobacterium abundance was associated with an increased proportion of CD4 + lymphocytes (p = 0.031) and M1 macrophages (p = 0.006), whilst M2 macrophages (p = 0.043) were under-represented in this cohort. Patients with increased Fusobacterium relative abundance in our mucinous CRC TCGA cohort tended to have better clinical outcomes (DSS: likelihood ratio p = 0.04, logrank p = 0.052). Fusobacterium abundance may be associated with improved outcomes in mucinous CRC, possibly due to a modulatory effect on the host immune response. Key messages: • Increased Fusobacterium relative abundance was not found to be associated with microsatellite instability in mucinous CRC. • Increased Fusobacterium relative abundance was associated with an M2/M1 macrophage switch, which is especially significant in mucinous CRC, where M2 macrophages are overexpressed. • Increased Fusobacterium relative abundance was associated with a significant improvement in disease specific survival in mucinous CRC. • Our findings were validated at a protein level within our own in house mucinous and non-mucinous rectal cancer cohorts.
  • Genomic analysis of two phlebotomine sand fly vectors of leishmania from the new and old World
    Labbe, Frederic; Abdeladhim, Maha; Abrudan, Jenica; Araki, Alejandra Saori; Araujo, Ricardo N.; Arensburger, Peter; Benoit, Joshua B.; Brazil, Reginaldo Pecanha; Bruno, Rafaela V.; Rivas, Gustavo Bueno da Silva D. S.; de Abreu, Vinicius Carvalho; Charamis, Jason; Coutinho-Abreu, Iliano V.; da Costa-Latge, Samara G.; Darby, Alistair; Dillon, Viv M.; Emrich, Scott J.; Fernandez-Medina, Daniela; Gontijo, Nelder Figueiredo; Flanley, Catherine M.; Gatherer, Derek; Genta, Fernando A.; Gesing, Sandra; Giraldo-Calderon, Gloria I.; Gomes, Bruno; Aguiar, Eric Roberto Guimaraes Rocha; Hamilton, James GC C.; Hamarsheh, Omar; Hawksworth, Mallory; Hendershot, Jacob M.; Hickner, Paul V.; Imler, Jean-Luc; Ioannidis, Panagiotis; Jennings, Emily C.; Kamhawi, Shaden; Karageorgiou, Charikleia; Kennedy, Ryan C.; Krueger, Andreas; Latorre-Estivalis, Jose M.; Ligoxygakis, Petros; Meireles-Filho, Antonio Carlos A.; Minx, Patrick; Miranda, Jose Carlos; Montague, Michael J.; Nowling, Ronald J.; Oliveira, Fabiano; Ortigao-Farias, Joao; Pavan, Marcio G.; Pereira, Marcos Horacio; Pitaluga, Andre Nobrega; Olmo, Roenick Proveti; Ramalho-Ortigao, Marcelo; Ribeiro, Jose MC C.; Rosendale, Andrew J.; Sant'Anna, Mauricio RV V.; Scherer, Steven E.; Secundino, Nagila FC C.; Shoue, Douglas A.; Moraes, Caroline da Silva D. S.; Gesto, Joao Silveira Moledo; Souza, Nataly Araujo; Syed, Zainulabueddin; Tadros, Samuel; Teles-de-Freitas, Rayane; Telleria, Erich L.; Tomlinson, Chad; Traub-Cseko, Yara M.; Marques, Joao Trindade; Tu, Zhijian; Unger, Maria F.; Valenzuela, Jesus; Ferreira, Flavia; de Oliveira, Karla PV V.; Vigoder, Felipe M.; Vontas, John; Wang, Lihui; Weedall, Gareth D.; Zhioua, Elyes; Richards, Stephen; Warren, Wesley C.; Waterhouse, Robert M.; Dillon, Rod J.; McDowell, Mary Ann (Public Library of Science, 2023-04-12)
    Phlebotomine sand flies are of global significance as important vectors of human disease, transmitting bacterial, viral, and protozoan pathogens, including the kinetoplastid parasites of the genus Leishmania, the causative agents of devastating diseases collectively termed leishmaniasis. More than 40 pathogenic Leishmania species are transmitted to humans by approximately 35 sand fly species in 98 countries with hundreds of millions of people at risk around the world. No approved efficacious vaccine exists for leishmaniasis and available therapeutic drugs are either toxic and/or expensive, or the parasites are becoming resistant to the more recently developed drugs. Therefore, sand fly and/or reservoir control are currently the most effective strategies to break transmission. To better understand the biology of sand flies, including the mechanisms involved in their vectorial capacity, insecticide resistance, and population structures we sequenced the genomes of two geographically widespread and important sand fly vector species: Phlebotomus papatasi, a vector of Leishmania parasites that cause cutaneous leishmaniasis, (distributed in Europe, the Middle East and North Africa) and Lutzomyia longipalpis, a vector of Leishmania parasites that cause visceral leishmaniasis (distributed across Central and South America). We categorized and curated genes involved in processes important to their roles as disease vectors, including chemosensation, blood feeding, circadian rhythm, immunity, and detoxification, as well as mobile genetic elements. We also defined gene orthology and observed micro-synteny among the genomes. Finally, we present the genetic diversity and population structure of these species in their respective geographical areas. These genomes will be a foundation on which to base future efforts to prevent vector-borne transmission of Leishmania parasites.
  • Nitrite reductase activity in F420-dependent sulphite reductase (Fsr) from Methanocaldococcus jannaschii
    Heryakusuma, Christian; Johnson, Eric F.; Purwantini, Endang; Mukhopadhyay, Biswarup (Microbiology Society, 2023-04-20)
    Methanocaldococcus jannaschii (Mj), a hyperthermophilic and evolutionarily deeply rooted methanogenic archaeon from a deep-sea hydrothermal vent, produces F420-dependent sulphite reductase (Fsr) in response to exposure to sulphite. This enzyme allows Mj to detoxify sulphite, a potent inhibitor of methyl coenzyme-M reductase (Mcr), by reducing it to sulphide with reduced coenzyme F420 (F420H2) as an electron donor; Mcr is essential for energy production for a methanogen. Fsr allows Mj to utilize sulphite as a sulphur source. Nitrite is another potent inhibitor of Mcr and is toxic to methanogens. It is reduced by most sulphite reductases. In this study, we report that MjFsr reduced nitrite to ammonia with F420H2 with physiologically relevant K m values (nitrite, 8.9 µM; F420H2, 9.7 µM). The enzyme also reduced hydroxylamine with a K m value of 112.4 µM, indicating that it was an intermediate in the reduction of nitrite to ammonia. These results open the possibility that Mj could use nitrite as a nitrogen source if it is provided at a low concentration of the type that occurs in its habitat.
  • Expression of anti-chikungunya single-domain antibodies in transgenic Aedes aegypti reduces vector competence for chikungunya virus and Mayaro virus
    Webb, Emily M.; Compton, Austin; Rai, Pallavi; Chuong, Christina; Paulson, Sally L.; Tu, Zhijian; Weger-Lucarelli, James (Frontiers, 2023-06-12)
    Chikungunya virus (CHIKV) and Mayaro virus (MAYV) are closely related alphaviruses that cause acute febrile illness accompanied by an incapacitating polyarthralgia that can persist for years following initial infection. In conjunction with sporadic outbreaks throughout the sub-tropical regions of the Americas, increased global travel to CHIKV- and MAYV-endemic areas has resulted in imported cases of MAYV, as well as imported cases and autochthonous transmission of CHIKV, within the United States and Europe. With increasing prevalence of CHIKV worldwide and MAYV throughout the Americas within the last decade, a heavy focus has been placed on control and prevention programs. To date, the most effective means of controlling the spread of these viruses is through mosquito control programs. However, current programs have limitations in their effectiveness; therefore, novel approaches are necessary to control the spread of these crippling pathogens and lessen their disease burden. We have previously identified and characterized an anti-CHIKV single-domain antibody (sdAb) that potently neutralizes several alphaviruses including Ross River virus and Mayaro virus. Given the close antigenic relationship between MAYV and CHIKV, we formulated a single defense strategy to combat both emerging arboviruses: we generated transgenic Aedes aegypti mosquitoes that express two camelid-derived anti-CHIKV sdAbs. Following an infectious bloodmeal, we observed significant reduction in CHIKV and MAYV replication and transmission potential in sdAb-expressing transgenic compared to wild-type mosquitoes; thus, this strategy provides a novel approach to controlling and preventing outbreaks of these pathogens that reduce quality of life throughout the tropical regions of the world.
  • charmm2gmx: An Automated Method to Port the CHARMM Additive Force Field to GROMACS
    Wacha, Andras F.; Lemkul, Justin A. (American Chemical Society, 2023-07-03)
    CHARMM is one of the most widely used biomolecular force fields. Although developed in close connection with a dedicated molecular simulation engine of the same name, it is also usable with other codes. GROMACS is a well-established, highly optimized, and multipurpose software for molecular dynamics, versatile enough to accommodate many different force field potential functions and the associated algorithms. Due to conceptional differences related to software design and the large amount of numeric data inherent to residue topologies and parameter sets, conversion from one software format to another is not straightforward. Here, we present an automated and validated means to port the CHARMM force field to a format read by the GROMACS engine, harmonizing the different capabilities of the two codes in a self-documenting and reproducible way with a bare minimum of user interaction required. Being based entirely on the upstream data files, the presented approach does not involve any hard-coded data, in contrast with previous attempts to solve the same problem. The heuristic approach used for perceiving the local internal geometry is directly applicable for analogous transformations of other force fields.
  • The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts
    Reinhold, Joanna M.; Halbert, Ella; Roark, Megan; Smith, Sierra N.; Stroh, Katherine M.; Siler, Cameron D.; McLeod, David S.; Lahondère, Chloé (2023-11-16)
    Background Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. Methods In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. Results We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. Conclusions This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.
  • Sex-differences in proteasome-dependent K48-polyubiquitin signaling in the amygdala are developmentally regulated in rats
    Farrell, Kayla; Auerbach, Aubrey; Liu, Catherine; Martin, Kiley; Pareno, Myasia; Ray, W. Keith; Helm, Richard F.; Biase, Fernando; Jarome, Timothy J. (2023-11-10)
    Background Sex differences have been observed in several brain regions for the molecular mechanisms involved in baseline (resting) and memory-related processes. The ubiquitin proteasome system (UPS) is a major protein degradation pathway in cells. Sex differences have been observed in lysine-48 (K48)-polyubiquitination, the canonical degradation mark of the UPS, both at baseline and during fear memory formation within the amygdala. Here, we investigated when, how, and why these baseline sex differences arise and whether both sexes require the K48-polyubiquitin mark for memory formation in the amygdala. Methods We used a combination of molecular, biochemical and proteomic approaches to examine global and protein-specific K48-polyubiquitination and DNA methylation levels at a major ubiquitin coding gene (Uba52) at baseline in the amygdala of male and female rats before and after puberty to determine if sex differences were developmentally regulated. We then used behavioral and genetic approaches to test the necessity of K48-polyubiquitination in the amygdala for fear memory formation. Results We observed developmentally regulated baseline differences in Uba52 methylation and total K48-polyubiquitination, with sexual maturity altering levels specifically in female rats. K48-polyubiquitination at specific proteins changed across development in both male and female rats, but sex differences were present regardless of age. Lastly, we found that genetic inhibition of K48-polyubiquitination in the amygdala of female, but not male, rats impaired fear memory formation. Conclusions These results suggest that K48-polyubiquitination differentially targets proteins in the amygdala in a sex-specific manner regardless of age. However, sexual maturity is important in the developmental regulation of K48-polyubiquitination levels in female rats. Consistent with these data, K48-polyubiquitin signaling in the amygdala is selectively required to form fear memories in female rats. Together, these data indicate that sex-differences in baseline K48-polyubiquitination within the amygdala are developmentally regulated, which could have important implications for better understanding sex-differences in molecular mechanisms involved in processes relevant to anxiety-related disorders such as post-traumatic stress disorder (PTSD).
  • Large Iodine Variability in Retail Cows' Milk in the U.S.: A Follow-Up Study among Different Retail Outlets
    Roseland, Janet M.; Phillips, Katherine M.; Vinyard, Bryan T.; Todorov, Todor; Ershow, Abby G.; Pehrsson, Pamela R. (MDPI, 2023-07-08)
    In a previous study, large variability in iodine content was found among samples of store brand retail milk at a single time point in a sampling taken from 24 nationwide U.S. locations for the USDA FoodData Central database, but the sampling plan was not designed to detect differences among locations. This follow-up study was carried out to evaluate iodine levels in retail milk across the U.S. over time. Milk samples (2% fat) were collected bimonthly in fourteen locations for one year and analyzed in duplicate. Control materials were used to support accuracy of results and ensure precision across analytical batches. The overall mean and standard error (SE) for iodine concentration were 82.5 (7.0) µg/240 mL serving, which was comparable to the previous national mean [85.0 (5.5) µg/240 mL]. A similar wide range among individual samples was detected (27.9–282 µg/240 mL). For some locations, the mean iodine concentration differed significantly from others, and differed from the national average by amounts ranging from −47 µg to +37 µg per serving. The between-sample range within location was large for some (up to 229 µg/serving) and minimal for others (as little as 13.2 µg/serving). These findings suggest iodine intake from some retail milk supplies could be over- or underestimated relative to the national average, even if the national average is suitable for population-wide intake estimates.
  • A large screen identifies beta-lactam antibiotics which can be repurposed to target the syphilis agent
    Hayes, Kathryn A.; Dressler, Jules M.; Norris, Steven J.; Edmondson, Diane G.; Jutras, Brandon L. (Springer Nature, 2023)
    Syphilis, caused by the spirochete Treponema pallidum subsp. pallidum (hereafter called T. pallidum), is re-emerging as a worldwide sexually transmitted infection. A single intramuscular dose of benzathine penicillin G is the preferred syphilis treatment option. Both supply shortage concerns and the potential for acquired antibiotic resistance further the need to broaden the repertoire of syphilis therapeutics. We reasoned that other β-lactams may be equally or more effective at targeting the disease-causing agent, Treponema pallidum, but have yet to be discovered due to a previous lack of a continuous in vitro culture system. Recent technical advances with respect to in vitro T. pallidum propagation allowed us to conduct a high-throughput screen of almost 100 β-lactams. Using several molecular and cellular approaches that we developed or adapted, we identified and confirmed the efficacy of several β-lactams that were similar to or outperformed the current standard, benzathine penicillin G. These options are either currently used to treat bacterial infections or are synthetic derivatives of naturally occurring compounds. Our studies not only identified additional potential therapeutics in the resolution of syphilis, but provide techniques to study the complex biology of T. pallidum— a spirochete that has plagued human health for centuries.
  • The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling
    Salar, Safoura; Ball, Nicolas E.; Baaziz, Hiba; Nix, Jay C.; Sobe, Richard C.; Compton, K. Karl; Zhulin, Igor B.; Brown, Anne M.; Scharf, Birgit E.; Schubot, Florian D. (Wiley, 2023-05)
    Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 angstrom resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 angstrom vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.
  • Key Factors Regulating the Interdomain Dynamics May Contribute to the Assembly of ASC
    Li, Tongtong; Gil Pineda, Laura I.; Stevens, Amy O.; He, Yi (MDPI, 2023-05-31)
    The canonical ASC domains, PYD and CARD, are interconnected by a lengthy, semi-flexible linker. The molecular basis and purpose of ASC’s highly dynamic feature remain elusive. In this study, all-atom molecular dynamics simulations were utilized to examine the role of the linker and the interdomain dynamics of the ASC monomer. As revealed in the principal component analysis (PCA), the flexible linker enables interdomain dynamics and rotation. The stumbling between domains is partially attributed to the helical portion of N-terminal residues in the linker. Additionally, the linker exhibits a certain structural preference due to the turn-type structural inclination of the N-terminal and the presence of several prolines on the linker. Such structural preferences lead to the unavailability of regions for PYD type I interactions to CARDs, as evidenced by the CARD spatial restraint analysis. In conclusion, the semi-flexible linker introduces functionally relevant interdomain dynamics, potentially enhancing PYD self-assembly and the subsequent assembly of the inflammasome complex.