28 results on '"Royce A. Wilkinson"'
Search Results
2. Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
- Author
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Andrew Santiago-Frangos, Laina N. Hall, Anna Nemudraia, Artem Nemudryi, Pushya Krishna, Tanner Wiegand, Royce A. Wilkinson, Deann T. Snyder, Jodi F. Hedges, Calvin Cicha, Helen H. Lee, Ava Graham, Mark A. Jutila, Matthew P. Taylor, and Blake Wiedenheft
- Subjects
CRISPR-Cas ,type III ,SARS-CoV-2 ,viral diagnostics ,CRISPR Dx ,COVID-19 ,Medicine (General) ,R5-920 - Abstract
Summary: There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min.
- Published
- 2021
- Full Text
- View/download PDF
3. A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric EpitheliumSummary
- Author
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Thomas A. Sebrell, Marziah Hashimi, Barkan Sidar, Royce A. Wilkinson, Liliya Kirpotina, Mark T. Quinn, Zeynep Malkoç, Paul J. Taylor, James N. Wilking, and Diane Bimczok
- Subjects
Diseases of the digestive system. Gastroenterology ,RC799-869 - Abstract
Background & Aims: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection. Keywords: Stomach, Organoid, Mononuclear Phagocyte, In Vitro Model
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- 2019
- Full Text
- View/download PDF
4. Protein-mediated genome folding allosterically enhances site-specific integration of foreign DNA into CRISPRs
- Author
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Andrew Santiago-Frangos, William S. Henriques, Tanner Wiegand, Colin C. Gauvin, Murat Buyukyoruk, Ava B. Graham, Royce A. Wilkinson, Lenny Triem, Kasahun Neselu, Edward T. Eng, Gabriel C. Lander, and Blake Wiedenheft
- Abstract
Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560 kDa integration complex structure that explains how Cas (Cas1-2/3) and non-Cas proteins (IHF) fold 150 base-pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3s rotate 100-degrees to expose DNA binding sites on either side of the Cas2 homodimer, that each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5’-GT.
- Published
- 2023
5. Intrinsic Signal Amplification by Type-III CRISPR-Cas Systems Provides a Sequence-Specific Viral Diagnostic
- Author
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Jodi F. Hedges, Laina N. Hall, Blake Wiedenheft, Deann T. Snyder, Tanner Wiegand, Anna Nemudraia, Pushya Krishna, Andrew Santiago-Frangos, Artem Nemudryi, Royce A. Wilkinson, Matthew P. Taylor, and Mark A. Jutila
- Subjects
biology ,Chemistry ,Oligonucleotide ,Specific detection ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,biology.protein ,CRISPR ,RNA ,Computational biology ,Signal amplification ,Polymerase ,Sequence (medicine) - Abstract
To combat viral pandemics, there is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here we repurposed the type III CRISPR-Cas system for sensitive and sequence specific detection of SARS-CoV-2 in an assay that can be performed in one hour or less. RNA recognition by type III systems triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons and cyclic oligonucleotides. We show that amplified products of the Cas10-polymerase are detectable using colorimetric or fluorometric readouts.
- Published
- 2020
6. AcrIF9 tethers non-sequence specific dsDNA to the CRISPR RNA-guided surveillance complex
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Royce A. Wilkinson, Wang-Ting Lu, Gabriel C. Lander, Sarah Golden, Andrew Santiago-Frangos, Marscha Hirschi, Alan R. Davidson, and Blake Wiedenheft
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Models, Molecular ,0301 basic medicine ,Protein Conformation ,Science ,General Physics and Astronomy ,Computational biology ,Plasma protein binding ,Proteus penneri ,Biochemistry ,Article ,General Biochemistry, Genetics and Molecular Biology ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Immune system ,Protein structure ,Bacterial Proteins ,CRISPR ,Bacteriophages ,Amino Acid Sequence ,lcsh:Science ,Peptide sequence ,Multidisciplinary ,Bacteria ,Sequence Homology, Amino Acid ,biology ,Chemistry ,Cryoelectron Microscopy ,RNA ,DNA ,General Chemistry ,biology.organism_classification ,030104 developmental biology ,Multiprotein Complexes ,Nucleic Acid Conformation ,lcsh:Q ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,Protein Binding ,RNA, Guide, Kinetoplastida - Abstract
Bacteria have evolved sophisticated adaptive immune systems, called CRISPR-Cas, that provide sequence-specific protection against phage infection. In turn, phages have evolved a broad spectrum of anti-CRISPRs that suppress these immune systems. Here we report structures of anti-CRISPR protein IF9 (AcrIF9) in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy). In addition to sterically blocking the hybridization of complementary dsDNA to the CRISPR RNA, our results show that AcrIF9 binding also promotes non-sequence-specific engagement with dsDNA, potentially sequestering the complex from target DNA. These findings highlight the versatility of anti-CRISPR mechanisms utilized by phages to suppress CRISPR-mediated immune systems., The anti-CRISPR protein IF9 (AcrIF9) specifically inhibits the type I-F CRISPR adaptive immune system. Here, the authors present the cryo-EM structure of AcrIF9 in complex with the type I-F CRISPR RNA-guided surveillance complex (Csy) and a dsDNA bound Csy-AcrIF9 structure, and find that AcrIF9 binding to the Csy complex triggers non-sequence specific dsDNA binding to Csy-AcrIF9, which might sequester the complex from its target DNA.
- Published
- 2020
7. Temporal detection and phylogenetic assessment of SARS-CoV-2 in municipal wastewater
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Blake Wiedenheft, Calvin Cicha, Murat Buyukyoruk, Tanner Wiegand, Kevin Surya, Karl K Vanderwood, Anna Nemudraia, Artem Nemudryi, and Royce A. Wilkinson
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Genetics ,Whole genome sequencing ,lcsh:R5-920 ,Phylogenetic tree ,SARS-CoV-2 ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,viruses ,fungi ,COVID-19 ,Biology ,Virology ,Article ,General Biochemistry, Genetics and Molecular Biology ,DNA sequencing ,wastewater-based epidemiology ,genome sequencing ,body regions ,Wastewater ,Report ,Time course ,lcsh:Medicine (General) ,skin and connective tissue diseases ,Genotyping ,Feces - Abstract
SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community., Graphical Abstract, Highlights SARS-CoV-2 RNA concentrations in wastewater correlate with COVID-19 epidemiology SARS-CoV-2 RNA levels in wastewater follow symptom onset by 5–8 days SARS-CoV-2 RNA levels in wastewater precede clinical PCR test results by 2–4 days SARS-CoV-2 genome from wastewater can trace phylogenetic origin, Nemudryi et al. demonstrate that wastewater can be used to monitor the progression and abatement of SARS-CoV-2 spread at the community level. The authors show a correlation between epidemiological indicators and viral concentrations measured in wastewater. In addition, they infer viral ancestry using a phylogenetic analysis of sequenced SARS-CoV-2 genome(s) from wastewater.
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- 2020
- Full Text
- View/download PDF
8. A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells
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M. Azim Surani, Aurélien J. Mazurie, Zhengyuan Wang, Meena Sukhwani, Ninuo Xia, Benjamin Angulo, Blake Wiedenheft, Charles C. Carey, Naoko Irie, Renee A. Reijo Pera, Kyle E. Orwig, Royce A. Wilkinson, Jun Cui, Fang Fang, Surani, Azim [0000-0002-8640-4318], and Apollo - University of Cambridge Repository
- Subjects
Male ,0301 basic medicine ,Time Factors ,Transcription, Genetic ,Somatic cell ,Cellular differentiation ,Human Embryonic Stem Cells ,Mice, Nude ,Biology ,Article ,Germline ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,SOX2 ,hemic and lymphatic diseases ,Testis ,PRDM1 ,medicine ,Animals ,Humans ,Induced pluripotent stem cell ,Gene Editing ,SOXB1 Transcription Factors ,PAX5 Transcription Factor ,Gene Expression Regulation, Developmental ,Cell Differentiation ,Cell Biology ,Spermatozoa ,Embryonic stem cell ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,embryonic structures ,Positive Regulatory Domain I-Binding Factor 1 ,Octamer Transcription Factor-3 ,030217 neurology & neurosurgery ,Germ cell ,Protein Binding ,Signal Transduction - Abstract
Dysregulation of genetic pathways during human germ cell development leads to infertility. Here, we analysed bona fide human primordial germ cells (hPGCs) to probe the developmental genetics of human germ cell specification and differentiation. We examined the distribution of OCT4 occupancy in hPGCs relative to human embryonic stem cells (hESCs). We demonstrated that development, from pluripotent stem cells to germ cells, is driven by switching partners with OCT4 from SOX2 to PAX5 and PRDM1. Gain- and loss-of-function studies revealed that PAX5 encodes a critical regulator of hPGC development. Moreover, an epistasis analysis indicated that PAX5 acts upstream of OCT4 and PRDM1. The PAX5-OCT4-PRDM1 proteins form a core transcriptional network that activates germline and represses somatic programmes during human germ cell differentiation. These findings illustrate the power of combined genome editing, cell differentiation and engraftment for probing human developmental genetics that have historically been difficult to study.
- Published
- 2018
9. Intrinsic signal amplification by type III CRISPR-Cas systems provides a sequence-specific SARS-CoV-2 diagnostic
- Author
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Anna Nemudraia, Mark A. Jutila, Pushya Krishna, Deann T. Snyder, Laina N. Hall, Calvin Cicha, Jodi F. Hedges, Matthew P. Taylor, Tanner Wiegand, Artem Nemudryi, Royce A. Wilkinson, Blake Wiedenheft, Helen H Lee, Ava Graham, and Andrew Santiago-Frangos
- Subjects
Medicine (General) ,type III ,viral diagnostics ,Computational biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,R5-920 ,Report ,Nasopharynx ,Humans ,CRISPR ,Guide RNA ,CRISPR-Cas ,Polymerase ,Sequence (medicine) ,Detection limit ,CRISPR Dx ,biology ,SARS-CoV-2 ,Chemistry ,Oligonucleotide ,COVID-19 ,RNA ,Molecular Diagnostic Techniques ,COVID-19 Nucleic Acid Testing ,biology.protein ,RNA, Viral ,Colorimetry ,CRISPR-Cas Systems ,point-of-care diagnostics ,Nucleic Acid Amplification Techniques ,RNA, Guide, Kinetoplastida - Abstract
There is an urgent need for inexpensive new technologies that enable fast, reliable, and scalable detection of viruses. Here, we repurpose the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2. RNA recognition by the type III CRISPR complex triggers Cas10-mediated polymerase activity, which simultaneously generates pyrophosphates, protons, and cyclic oligonucleotides. We show that all three Cas10-polymerase products are detectable using colorimetric or fluorometric readouts. We design ten guide RNAs that target conserved regions of SARS-CoV-2 genomes. Multiplexing improves the sensitivity of amplification-free RNA detection from 107 copies/μL for a single guide RNA to 106 copies/μL for ten guides. To decrease the limit of detection to levels that are clinically relevant, we developed a two-pot reaction consisting of RT-LAMP followed by T7-transcription and type III CRISPR-based detection. The two-pot reaction has a sensitivity of 200 copies/μL and is completed using patient samples in less than 30 min., Graphical abstract, Recognition of a complementary target RNA by the type III CRISPR systems uniquely triggers the activation of a CRISPR-associated polymerase domain in Cas10. The polymerase generates oligoadenylates, protons, and pyrophosphates. Santiago-Frangos et al. repurposed the type III CRISPR-Cas system for sensitive and sequence-specific detection of SARS-CoV-2 by developing three different detection methods that rely on each of these products.
- Published
- 2021
10. Live imaging analysis of human gastric epithelial spheroids reveals spontaneous rupture, rotation, and fusion events
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Brian A. Perrino, Blake Wiedenheft, Barkan Sidar, Linda C. Samuelson, James N. Wilking, Diane Bimczok, Rachel Bruns, Andrew Sebrell, Royce A. Wilkinson, and Paul J. Taylor
- Subjects
0301 basic medicine ,Spontaneous rupture ,Adult ,Male ,Pathology ,medicine.medical_specialty ,Histology ,Rotation ,Matrix (biology) ,Biology ,Membrane Fusion ,Article ,Pathology and Forensic Medicine ,Cell Fusion ,03 medical and health sciences ,Imaging, Three-Dimensional ,Live cell imaging ,Spheroids, Cellular ,Organoid ,medicine ,Humans ,Cell Proliferation ,Rupture ,Matrigel ,Wound Healing ,Rupture, Spontaneous ,Stomach ,Spheroid ,Epithelial Cells ,Cell Biology ,Middle Aged ,Epithelium ,Imaging analysis ,Organoids ,Drug Combinations ,030104 developmental biology ,medicine.anatomical_structure ,Phenotype ,embryonic structures ,Female ,Proteoglycans ,Collagen ,Laminin - Abstract
Three-dimensional cultures of primary epithelial cells including organoids, enteroids and epithelial spheroids have become increasingly popular for studies of gastrointestinal development, mucosal immunology and epithelial infection. However, little is known about the behavior of these complex cultures in their three-dimensional culture matrix. Therefore, we performed extended time-lapse imaging analysis (up to 4 days) of human gastric epithelial spheroids generated from adult tissue samples in order to visualize the dynamics of the spheroids in detail. Human gastric epithelial spheroids cultured in our laboratory grew to an average diameter of 443.9 ± 34.6 µm after 12 days, with the largest spheroids reaching diameters of > 1,000 µm. Live imaging analysis revealed that spheroid growth was associated with cyclic rupture of the epithelial shell at a frequency of 0.32 ± 0.1/day, which led to the release of luminal contents. Spheroid rupture usually resulted in an initial collapse, followed by spontaneous re-formation of the spheres. Moreover, spheroids frequently rotated around their axes within the Matrigel matrix, possibly propelled by basolateral pseudopodia-like formations of the epithelial cells. Interestingly, adjacent spheroids occasionally underwent luminal fusion, as visualized by injection of individual spheroids with FITC-dextran (40 kDa). In summary, our analysis revealed unexpected dynamics in human gastric spheroids that challenge our current view of cultured epithelia as static entities and that may need to be considered when performing spheroid infection experiments.
- Published
- 2017
11. Cas1 and the Csy complex are opposing regulators of Cas2/3 nuclease activity
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Joshua Carter, Saikat Chowdhury, MaryClare F. Rollins, Blake Wiedenheft, Royce A. Wilkinson, Sarah Golden, Gabriel C. Lander, and Joseph Bondy-Denomy
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0301 basic medicine ,1.1 Normal biological development and functioning ,Biology ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Multienzyme Complexes ,Cas1 ,Underpinning research ,Genetics ,CRISPR ,Gene ,Trans-activating crRNA ,Nuclease ,Multidisciplinary ,Deoxyribonucleases ,RNA ,Cas ,Acquired immune system ,type I-F ,Cell biology ,030104 developmental biology ,Emerging Infectious Diseases ,PNAS Plus ,chemistry ,Pseudomonas aeruginosa ,biology.protein ,CRISPR Loci ,CRISPR-Cas Systems ,Cas2/3 ,DNA - Abstract
The type I-F CRISPR adaptive immune system in Pseudomonas aeruginosa (PA14) consists of two CRISPR loci and six CRISPR-associated (cas) genes. Type I-F systems rely on a CRISPR RNA (crRNA)-guided surveillance complex (Csy complex) to bind foreign DNA and recruit a trans-acting nuclease (i.e., Cas2/3) for target degradation. In most type I systems, Cas2 and Cas3 are separate proteins involved in adaptation and interference, respectively. However, in I-F systems, these proteins are fused into a single polypeptide. Here we use biochemical and structural methods to show that two molecules of Cas2/3 assemble with four molecules of Cas1 (Cas2/32:Cas14) into a four-lobed propeller-shaped structure, where the two Cas2 domains form a central hub (twofold axis of symmetry) flanked by two Cas1 lobes and two Cas3 lobes. We show that the Cas1 subunits repress Cas2/3 nuclease activity and that foreign DNA recognition by the Csy complex activates Cas2/3, resulting in bidirectional degradation of DNA targets. Collectively, this work provides a structure of the Cas1–2/3 complex and explains how Cas1 and the target-bound Csy complex play opposing roles in the regulation of Cas2/3 nuclease activity.
- Published
- 2017
12. A Novel Gastric Spheroid Co-culture Model Reveals Chemokine-Dependent Recruitment of Human Dendritic Cells to the Gastric Epithelium
- Author
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Barkan Sidar, Marziah Hashimi, Thomas A. Sebrell, Paul J. Taylor, James N. Wilking, Diane Bimczok, Zeynep Malkoc, Royce A. Wilkinson, Liliya N. Kirpotina, and Mark T. Quinn
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Organoid ,MoDC, monocyte-derived dendritic cell ,RT-PCR, reverse-transcription polymerase chain reaction ,0301 basic medicine ,Chemokine ,MNP, mononuclear phagocyte ,Monocytes ,0302 clinical medicine ,In Vitro Model ,ADC, dendritic cell ,Cells, Cultured ,Original Research ,GFP, green fluorescent protein ,Stomach ,Gastroenterology ,3. Good health ,Organoids ,Immunosurveillance ,CXCL1 ,Editorial ,medicine.anatomical_structure ,030211 gastroenterology & hepatology ,FITC, fluorescein isothiocyanate ,Chemokines ,TLR, Toll-like receptor ,Biology ,Helicobacter Infections ,03 medical and health sciences ,Spheroids, Cellular ,medicine ,Humans ,lcsh:RC799-869 ,Mononuclear Phagocyte ,CXCL16 ,CXCL17 ,Helicobacter pylori ,Hepatology ,Gene Expression Profiling ,Epithelial Cells ,Dendritic Cells ,biology.organism_classification ,Coculture Techniques ,digestive system diseases ,IL, interleukin ,CagA, cytotoxin-associated antigen ,CCL20 ,030104 developmental biology ,Gene Expression Regulation ,Gastric Mucosa ,biology.protein ,Cancer research ,lcsh:Diseases of the digestive system. Gastroenterology - Abstract
Background & Aims: Gastric dendritic cells (DCs) control the adaptive response to infection with Helicobacter pylori, a major risk factor for peptic ulcer disease and gastric cancer. We hypothesize that DC interactions with the gastric epithelium position gastric DCs for uptake of luminal H pylori and promote DC responses to epithelial-derived mediators. The aim of this study was to determine whether the gastric epithelium actively recruits DCs using a novel co-culture model of human gastric epithelial spheroids and monocyte-derived DCs. Methods: Spheroid cultures of primary gastric epithelial cells were infected with H pylori by microinjection. Co-cultures were established by adding human monocyte-derived DCs to the spheroid cultures and were analyzed for DC recruitment and antigen uptake by confocal microscopy. Protein array, gene expression polymerase chain reaction array, and chemotaxis assays were used to identify epithelial-derived chemotactic factors that attract DCs. Data from the co-culture model were confirmed using human gastric tissue samples. Results: Human monocyte-derived DCs co-cultured with gastric spheroids spontaneously migrated to the gastric epithelium, established tight interactions with the epithelial cells, and phagocytosed luminally applied H pylori. DC recruitment was increased upon H pylori infection of the spheroids and involved the activity of multiple chemokines including CXCL1, CXCL16, CXCL17, and CCL20. Enhanced chemokine expression and DC recruitment to the gastric epithelium also was observed in H pylori–infected human gastric tissue samples. Conclusions: Our results indicate that the gastric epithelium actively recruits DCs for immunosurveillance and pathogen sampling through chemokine-dependent mechanisms, with increased recruitment upon active H pylori infection. Keywords: Stomach, Organoid, Mononuclear Phagocyte, In Vitro Model
- Published
- 2019
13. Improved guanide compounds which bind the CXCR4 co-receptor and inhibit HIV-1 infection
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Joyce B. Shepard, Alan J. Weaver, Kejing Song, Mohamed E. Labib, Martin Teintze, Seth H. Pincus, and Royce A. Wilkinson
- Subjects
Receptors, CXCR4 ,Co-receptor ,Anti-HIV Agents ,Clinical Biochemistry ,Biguanides ,Pharmaceutical Science ,Spermine ,HIV Infections ,Microbial Sensitivity Tests ,Biochemistry ,Article ,Cell Line ,Structure-Activity Relationship ,chemistry.chemical_compound ,Drug Discovery ,Humans ,Structure–activity relationship ,Receptor ,Molecular Biology ,Cell Proliferation ,Dose-Response Relationship, Drug ,Molecular Structure ,Cell growth ,Organic Chemistry ,Cell migration ,Spermidine ,chemistry ,Cell culture ,HIV-1 ,Molecular Medicine - Abstract
The G-protein coupled receptor CXCR4 is a co-receptor for HIV-1 infection and is involved in signaling cell migration and proliferation. In a previous study of non-peptide, guanide-based CXCR4-binding compounds, spermine and spermidine phenylguanides inhibited HIV-1 entry at low micromolar concentrations. Subsequently, crystal structures of CXCR4 were used to dock a series of naphthylguanide derivatives of the polyamines spermidine and spermine. Synthesis and evaluation of the naphthylguanide compounds identified our best compound, spermine tris-1-naphthylguanide, which bound CXCR4 with an IC(50) of 40 nM and inhibited the infection of TZM-bl cells with X4, but not R5, strains of HIV-1 with an IC(50) of 50-100 nM.
- Published
- 2013
14. Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System
- Author
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Shefah Qazi, Trevor Douglas, Kimberly McCoy, Blake Wiedenheft, Royce A. Wilkinson, and Heini M. Miettinen
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0301 basic medicine ,Scaffold protein ,viruses ,Pharmaceutical Science ,02 engineering and technology ,Article ,Genome engineering ,03 medical and health sciences ,Endonuclease ,Drug Delivery Systems ,Genome editing ,Bacterial Proteins ,CRISPR-Associated Protein 9 ,Drug Discovery ,CRISPR ,Humans ,Clustered Regularly Interspaced Short Palindromic Repeats ,Bacteriophage P22 ,Genetics ,Drug Carriers ,biology ,Cas9 ,021001 nanoscience & nanotechnology ,Endonucleases ,Cell biology ,030104 developmental biology ,Capsid ,biology.protein ,Molecular Medicine ,Nanoparticles ,RNA Editing ,Nanocarriers ,0210 nano-technology ,Genetic Engineering - Abstract
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.
- Published
- 2016
15. Novel Compounds Containing Multiple Guanide Groups That Bind the HIV Coreceptor CXCR4
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Joyce B. Shepard, Sarah K. Walton, Edward P. Bergin, Mohamed E. Labib, Martin Teintze, Royce A. Wilkinson, and Seth H. Pincus
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Pharmacology ,chemistry.chemical_classification ,Receptors, CXCR4 ,Arginine ,Anti-HIV Agents ,Spermine ,Peptide ,Biology ,Antiviral Agents ,Cell Line ,Spermidine ,chemistry.chemical_compound ,Infectious Diseases ,Biochemistry ,chemistry ,Cell culture ,HIV-1 ,Humans ,Pharmacology (medical) ,Peptides ,Receptor ,Cytotoxicity ,Maraviroc - Abstract
The G-protein-coupled receptor CXCR4 acts as a coreceptor for human immunodeficiency virus type 1 (HIV-1) infection, as well as being involved in signaling cell migration and proliferation. Compounds that block CXCR4 interactions have potential uses as HIV entry inhibitors to complement drugs such as maraviroc that block the alternate coreceptor CCR5 or in cancer therapy. The peptide T140, which contains five arginine residues, is the most potent antagonist of CXCR4 developed to date. In a search for nonpeptide CXCR4 ligands that could inhibit HIV entry, three series of compounds were synthesized from 12 linear and branched polyamines with 2, 3, 4, 6, or 8 amino groups, which were substituted to produce the corresponding guanidines, biguanides, or phenylguanides. The resulting compounds were tested for their ability to compete with T140 for binding to the human CXCR4 receptor expressed on mammalian cells. The most effective compounds bound CXCR4 with a 50% inhibitory concentration of 200 nM, and all of the compounds had very low cytotoxicity. Two series of compounds were then tested for their ability to inhibit the infection of TZM-bl cells with X4 and R5 strains of HIV-1. Spermine phenylguanide and spermidine phenylguanide inhibited infection by X4 strains, but not by R5 strains, at low micromolar concentrations. These results support further investigation and development of these compounds as HIV entry inhibitors.
- Published
- 2011
16. Peptides Selected from a Phage Display Library with an HIV-Neutralizing Antibody Elicit Antibodies to HIV gp120 in Rabbits, But Not to the Same Epitope
- Author
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Royce A. Wilkinson, James B. Burritt, Jon M. Jacobs, Martin Teintze, Abraham Pinter, Dustin Slunaker, Jody R. Evans, Charles A. Parkos, and Seth H. Pincus
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Phage display ,medicine.drug_class ,viruses ,Immunology ,HIV Envelope Protein gp120 ,Antibodies, Viral ,Monoclonal antibody ,Epitope ,Epitopes ,Neutralization Tests ,Peptide Library ,Virology ,medicine ,Animals ,Neutralizing antibody ,Peptide library ,Peptide sequence ,AIDS Vaccines ,biology ,Antibodies, Monoclonal ,virus diseases ,HIV envelope protein ,Molecular biology ,Infectious Diseases ,Epitope mapping ,biology.protein ,Rabbits ,Peptides ,Epitope Mapping ,Protein Binding - Abstract
Monoclonal antibodies specific for the conserved CD4 binding site region of the HIV envelope protein gp120 were used to select phage from two different random peptide display libraries. Synthetic peptides were made with sequences corresponding to those displayed on the selected phage, and peptide-protein fusions were expressed that contained the selected phage-displayed peptide sequence and either the N-terminal domain of the phage pIII protein or the small heat shock protein of Methanococcus jannaschii or both. For monoclonal antibody 5145A, these constructs containing the selected peptide sequences were all capable of specifically inhibiting the binding of 5145A to HIV-1 gp120. Rabbits immunized with peptide-protein fusions produced antisera that bound to recombinant HIV-1 gp120, but did not bind to HIV-infected cells nor neutralize HIV. The antisera also did not compete with CD4 or antibodies to the CD4 binding site for binding to gp120.
- Published
- 2007
17. Structure of the Fab Fragment of F105, a Broadly Reactive Anti-Human Immunodeficiency Virus (HIV) Antibody That Recognizes the CD4 Binding Site of HIV Type 1 gp120
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Chayne L. Piscitelli, C. Martin Lawrence, Martin Teintze, Marshall R. Posner, Lisa A. Cavacini, and Royce A. Wilkinson
- Subjects
Models, Molecular ,Conformational change ,medicine.drug_class ,Molecular Sequence Data ,Immunology ,HIV Antibodies ,HIV Envelope Protein gp120 ,medicine.disease_cause ,Monoclonal antibody ,Microbiology ,Virus ,Epitope ,Immunoglobulin Fab Fragments ,Antibody Specificity ,Virology ,medicine ,Amino Acid Sequence ,Binding site ,Crystallography ,biology ,Lipid bilayer fusion ,Simian immunodeficiency virus ,Complementarity Determining Regions ,Insect Science ,CD4 Antigens ,HIV-1 ,biology.protein ,Pathogenesis and Immunity ,Antibody - Abstract
A key step in the development of a successful vaccine against human immunodeficiency virus (HIV) will be the design of immunogens capable of generating an effective humoral immune response (3). Antibodies with two features characterize such a response. They must show, at the same time, both potency and broad specificity. A number of human antibodies that recognize elements of the conserved CD4 binding site of HIV type 1 (HIV-1) gp120 have now been isolated from HIV-infected patients (2, 11, 16, 24, 38, 40, 47, 49, 59, 65). This includes immunoglobulin G1 (IgG1) b12 (38, 47, 53), one of the most potent and broadly reactive anti-HIV antibodies known. In contrast, other CD4 binding-site antibodies are less potent towards many clinical isolates, even though they may be broadly cross-reactive. F105, the subject of this paper, is representative of the latter group. F105 is an IgG1 κ human monoclonal antibody isolated from an HIV-infected individual (49). It binds to the CD4 binding sites of both trimeric and monomeric gp120 and is capable of neutralizing various strains of HIV (e.g., IIIB [HXBc2], MN, RF, and SF2) (7, 49, 58) but is less successful against many primary clinical isolates (12, 32). F105 did not show evidence of anti-HIV-1 activity or a viral load decrease in a phase I dose-escalation study (5, 70). However, in triple and quadruple combination therapies with anti-HIV monoclonal antibodies (2F5, 2G12, and 694/98D) with other specificities, a complete and synergistic neutralization of the SHIV-Vpu+ chimeric simian-human immunodeficiency virus was seen in macaque peripheral blood mononuclear cells in vitro and in an in vivo macaque model that mimics mucosal exposure during intrapartum virus transmission (1, 31). Crystallographic studies of the ternary complex of the HIV gp120 core, CD4, and antibody 17b provided the first look at the structure of gp120 and its interactions with CD4 (26-28, 65). CD4 was found to bind at the nexus of the inner domain, the outer domain, and the bridging sheet of gp120 (26, 27). A large body of biochemical and biophysical data indicates a considerable conformational change in gp120 upon binding to CD4 (4, 6, 15, 24, 26, 27, 39, 41, 50, 55, 60, 62-69, 72, 73). The conformational change results in the formation and/or exposure of the chemokine receptor sites (62, 64), thus promoting further viral attachment and membrane fusion. The molecular reorganization that results upon binding of CD4 is revealed by the structure of an unliganded simian immunodeficiency virus (SIV) gp120 core (6). With a few important exceptions, the structure of the outer domain is quite similar to that seen in the CD4-bound state. In contrast, the structure of the inner domain is markedly different. A comparison of CD4-bound and unliganded gp120 shows that the conformational change is not a simple movement of the inner domain as a rigid body. Rather, the inner domain is comprised of a set of distinct substructures that move relatively independently of one another (6). The binding of CD4 results in a rearrangement of these secondary structural elements within the inner domain (6). As predicted (24), the bridging sheet is not present in the structure of unliganded gp120 (6). The structure of antibody b12 was determined by Saphire et al. (52-54). The structure revealed an extended CDR H3 loop with an apical tryptophan residue that is thought to recognize the Phe43 binding pocket of gp120 (53, 75). This putative interaction places significant constraints on possible gp120/b12 interactions, allowing an interaction between b12 and the CD4-bound conformation of gp120 to be modeled (53, 75). This model suggests that the broad neutralizing activity of b12 lies in its ability to interact with conserved features of the CD4 binding site through recognition of the Phe43 pocket and interactions with main chain atoms of gp120 and in its ability to recognize trimeric gp120 on the native viral surface. A further understanding of the molecular properties that confer broad reactivity and potency upon a CD4 binding-site antibody are of substantial interest. The underlying principles are likely to impact the design of new immunogens with the potential to elicit a successful immune response. In this regard, the structure of F105 provides an opportunity to compare and contrast the structure of a broadly reactive but nonpotent CD4 binding-site antibody (F105) with that of a broadly neutralizing antibody with an overlapping epitope (b12). The comparison provides significant insight into the molecular properties that impart broad reactivity and potency upon a CD4 binding-site antibody.
- Published
- 2005
18. Immunological Characteristics Associated with the Protective Efficacy of Antibodies to Ricin
- Author
-
Leta Eng, Audrey V. Stout, Corrie L. Cooke, Seth H. Pincus, Massimo Maddaloni, and Royce A. Wilkinson
- Subjects
Immunology ,Antibody Affinity ,Ricin ,medicine.disease_cause ,Cell Line ,Mice ,chemistry.chemical_compound ,Antibody Specificity ,In vivo ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Immunology and Allergy ,Avidity ,Enzyme Inhibitors ,Cytotoxicity ,Mice, Inbred BALB C ,Hybridomas ,biology ,Cytotoxins ,Toxin ,Antibodies, Monoclonal ,Molecular biology ,Isotype ,In vitro ,Enzyme Activation ,Protein Subunits ,chemistry ,biology.protein ,Binding Sites, Antibody ,Antibody - Abstract
A/B toxins, produced by bacteria and plants, are among the deadliest molecules known. The B chain binds the cell, whereas the A chain exerts the toxic effect. Both anti-A chain and anti-B chain Abs can neutralize toxins in vivo and in vitro. B chain Abs block binding of the toxin to the cell. It is not known how anti-A chain Abs function. Working with ricin toxin, we demonstrate that immunization with A chain induces greater protection than immunization with B chain. A panel of mAbs, binding to A chain, B chain, or both chains, has been produced and characterized. Immunologic characteristics evaluated include isotype, relative avidity, and epitope specificity. The ability to inhibit ricin enzymatic or cell binding activity was studied, as was the ability to block ricin-mediated cellular cytotoxicity on human and murine cell lines. Finally, the in vivo protective efficacy of the Abs in mice was studied. The Ab providing the greatest in vivo protective efficacy was directed against the A chain. It had the greatest relative avidity and the greatest ability to block enzymatic function and neutralize cytotoxicity. Interestingly, we also obtained an anti-A chain Ab that bound with high avidity, blocked enzymatic activity, did not neutralize cytotoxicity, and actually enhanced the in vivo toxicity of ricin. Anti-A chain Abs with moderate avidity had no in vivo effect, nor did any anti-B chain Abs.
- Published
- 2004
19. A Modified SCID Mouse Model of HIV Infection with Utility for Testing Anti-HIV Therapies
- Author
-
Royce A. Wilkinson, Tamera K. Marcotte, Seth H. Pincus, William C. Olson, and Hua Fang
- Subjects
Anti-HIV Agents ,T cell ,Immunology ,HIV Infections ,Spleen ,Mice, SCID ,Antibodies, Viral ,Virus ,Mice ,Mice, Inbred NOD ,Virology ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,NOD mice ,biology ,medicine.disease ,biology.organism_classification ,Lymphoma ,Disease Models, Animal ,Infectious Diseases ,medicine.anatomical_structure ,Lentivirus ,HIV-1 ,biology.protein ,Viral disease ,Antibody ,CD4 Immunoadhesins - Abstract
Using human tumor cells we have developed a mouse model of active HIV infection that may be used for testing antiviral agents, although it does not reflect the pathogenesis of human infection. Irradiated SCID/NOD mice are injected with a tumor of human CD4+ lymphoma cells susceptible to infection and at a separate site, tumor cells persistently infected with either primary or T cell line-adapted strains of HIV. The spread of infection from the infected to the susceptible tumor is monitored as plasma p24 and the presence of HIV-infected cells in the spleen. We have used this model to examine the relative efficacy of neutralizing anti-HIV antibodies to halt the spread of infection. We have found that the tetrameric CD4-antibody fusion protein, CD4-IgG2, is highly effective compared to an anti-V3 loop antibody. This animal model, while not replicating the human disease, allows for the simultaneous testing of efficacy, toxicity, and pharmacokinetics of potential new antiviral therapies. The model can easily be powered to enable comparisons between different therapeutic agents and dosing regimens.
- Published
- 2003
20. A CRISPR method for genome engineering
- Author
-
Royce A. Wilkinson and Blake Wiedenheft
- Subjects
Cell type ,Multicellular organism ,biology ,CRISPR ,General Medicine ,Computational biology ,Review Article ,Bioinformatics ,biology.organism_classification ,Bacteria ,Archaea ,Genome engineering - Abstract
Clustered regularly interspaced short palindromic repeat (CRISPR) RNA-guided adaptive immune systems that protect bacteria and archaea from infection by viruses are now being routinely repurposed for genome engineering in a wide variety of cell types and multicellular organisms.
- Published
- 2014
21. Antibacterial activity of THAM Trisphenylguanide against methicillin-resistant Staphylococcus aureus
- Author
-
Sarah K. Walton, Joyce B. Shepard, Thomas J. Wright, Jovanka M. Voyich, Amanda R. Radke, Mohamed E. Labib, Royce A. Wilkinson, Alan J. Weaver, Robert L. Watkins, Milat B. Awel, Elizabeth Erikson, Martin Teintze, and Catherine Cooper
- Subjects
Acinetobacter baumannii ,Bacterial Diseases ,Applied Microbiology ,Staphylococcus ,Antibiotics ,Biguanides ,lcsh:Medicine ,medicine.disease_cause ,Guanidines ,Biochemistry ,Mice ,Drug Discovery ,Enterococcus faecalis ,Medicine and Health Sciences ,lcsh:Science ,0303 health sciences ,Multidisciplinary ,biology ,Molecular Structure ,Animal Models ,Medical microbiology ,Antimicrobial ,3. Good health ,Anti-Bacterial Agents ,Infectious Diseases ,Staphylococcus aureus ,Pseudomonas aeruginosa ,Antibacterial activity ,Research Article ,Biotechnology ,Methicillin-Resistant Staphylococcus aureus ,Drug Research and Development ,Infectious Disease Control ,medicine.drug_class ,Mouse Models ,Microbial Sensitivity Tests ,Research and Analysis Methods ,Microbiology ,03 medical and health sciences ,Minimum inhibitory concentration ,Model Organisms ,Microbial Control ,medicine ,Escherichia coli ,Animals ,030304 developmental biology ,Pharmacology ,Biology and life sciences ,030306 microbiology ,lcsh:R ,biochemical phenomena, metabolism, and nutrition ,biology.organism_classification ,bacterial infections and mycoses ,Methicillin-resistant Staphylococcus aureus ,Microbial pathogens ,Small Molecules ,lcsh:Q ,Bacterial pathogens - Abstract
This study investigated the potential antibacterial activity of three series of compounds synthesized from 12 linear and branched polyamines with 2–8 amino groups, which were substituted to produce the corresponding guanides, biguanides, or phenylguanides, against Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Antibacterial activity was measured for each compound by determining the minimum inhibitory concentration against the bacteria, and the toxicity towards mammalian cells was determined. The most effective compound, THAM trisphenylguanide, was studied in time-to-kill and cytoplasmic leakage assays against methicillin-resistant Staphylococcus aureus (MRSA, USA300) in comparison to chlorhexidine. Preliminary toxicity and MRSA challenge studies in mice were also conducted on this compound. THAM trisphenylguanide showed significant antibacterial activity (MIC ~1 mg/L) and selectivity against MRSA relative to all the other bacteria examined. In time-to-kill assays it showed increased antimicrobial activity against MRSA versus chlorhexidine. It induced leakage of cytoplasmic content at concentrations that did not reduce cell viability, suggesting the mechanism of action may involve membrane disruption. Using an intraperitoneal mouse model of invasive MRSA disease, THAM trisphenylguanide reduced bacterial burden locally and in deeper tissues. This study has identified a novel guanide compound with selective microbicidal activity against Staphylococcus aureus, including a methicillin-resistant (MRSA) strain.
- Published
- 2013
22. Approaches to the Syntheses of 2- and 3-Phosphonomethyl Derivatives of Arabinose Via Pentacovalent Oxaphospholene Methodology
- Author
-
Todd A. Madsen, Todd R. Boehlow, Larry A. Alegria, Cynthia K. McClure, and Royce A. Wilkinson
- Subjects
Arabinose ,Stereochemistry ,Organic Chemistry ,Diol ,Biochemistry ,Inorganic Chemistry ,chemistry.chemical_compound ,chemistry ,Aldol reaction ,Derivative (finance) ,Glyceraldehyde acetonide ,Glyoxal ,Organic chemistry ,Stereoselectivity - Abstract
Approaches to the syntheses of 2- and 3-phosphonomethyl derivatives of arabinose via pentacovalent oxaphospholene methodology are outlined. Condensation of the requisite pentacovalent oxaphospholenes with either a mono-protected glyoxal derivative or glyceraldehyde acetonide were highly stereoselective, producing the syn aldol product as the major isomer in both cases. The reduction of 4a to the diol did not give the expected stereoselectivity. Stereochemical correlation of 5 and 6 were via the carbonates.
- Published
- 1999
23. Novel guanide-substituted compounds bind to CXCR4 and inhibit breast cancer metastasis
- Author
-
Jean R. Starkey, Royce A. Wilkinson, Joyce B. Shepard, and Martin Teintze
- Subjects
Oncology ,Cancer Research ,medicine.medical_specialty ,Receptors, CXCR4 ,Lung Neoplasms ,medicine.drug_class ,Biguanides ,Breast Neoplasms ,Mice, SCID ,CXCR4 ,Guanidines ,Metastasis ,Mice ,Breast cancer ,Dogs ,Internal medicine ,Cell Line, Tumor ,medicine ,Animals ,Humans ,Pharmacology (medical) ,Neoplasm Invasiveness ,Receptor ,Pharmacology ,Chemistry ,Biguanide ,medicine.disease ,Chemokine CXCL12 ,Cell culture ,Toxicity ,Cancer cell ,Cancer research ,Female ,Drug Screening Assays, Antitumor ,Neoplasm Transplantation ,Protein Binding - Abstract
CXCR4 has been shown to be overexpressed on breast cancer cells including the human MDA-MB-231 cell line. Cancer cells overexpressing the CXCR4 receptor are capable of undergoing metastasis to organs expressing high levels of CXCL12. We have synthesized numerous guanide, biguanide, phenylguanide, and naphthylguanide compounds that bind to CXCR4 at the CXCL12-binding site and thus should prevent CXCR4-facilitated cancer metastasis. The novel compounds presented here were tested for CXCR4 affinity, toxicity, receptor activation, and for their ability to prevent breast cancer metastases. Three of the compounds bound to CXCR4 at IC50 values of 0.06-0.2 μmol/l, with no associated cell toxicity or receptor activation at these concentrations. These high CXCR4 affinity compounds also showed inhibition of in-vitro wound migration. They were then tested in an in-vivo mouse breast cancer lung colony model. All of these compounds showed reductions in the number of MDA-MB-231 lung metastases compared with mock-treated control mice without evidence of cardiac, liver, or kidney toxicity in treated mice.
- Published
- 2013
24. Sphaeric Acid, a New Succinic Acid Derivative from a Sphaeropsis sp
- Author
-
Andrea A. Stierle, Royce A. Wilkinson, and Gary A. Strobel
- Subjects
Pharmacology ,chemistry.chemical_classification ,Stereochemistry ,Organic Chemistry ,Diol ,Pharmaceutical Science ,Biology ,Analytical Chemistry ,chemistry.chemical_compound ,Transformation (genetics) ,Sphaeric acid ,Dicarboxylic acid ,Complementary and alternative medicine ,chemistry ,Succinic acid ,Drug Discovery ,Molecular Medicine ,Aliphatic compound ,Fermentation broth ,Derivative (chemistry) - Abstract
The structure of sphaeric acid (1), a novel succinic acid derivative isolated from the fermentation broth of a Sphaeropsis sp., was determined by spectral data and synthetic transformation to the diol of sphaeric acid and subsequently to a pair of γ-lactones (2 and 3).
- Published
- 1998
25. Anti-HIV Immunotoxins
- Author
-
Royce A. Wilkinson, Seth H. Pincus, and Hua Fang
- Subjects
Pokeweed antiviral protein ,Antigen ,business.industry ,Anti hiv ,Immunotoxin ,Infected cell ,Immunology ,Human immunodeficiency virus (HIV) ,Medicine ,business ,medicine.disease_cause ,Clearance - Abstract
The use of targeted therapies to treat infectious diseases is a novel application that requires a persistent infection that cannot be cleared by other means and the expression of microbial antigens on the surface of productively-infected cells. Although some bacterial or parasitic infections may meet these criteria, chronic viral infections and microbe-induced neoplasia are the most likely targets. In this chapter we will discuss the use of immunotoxins to treat human immunodeficiency virus (HIV) infection. The results demonstrate the importance of targeting the appropriate molecules and utilizing the cell biology of the microorganism.
- Published
- 2002
26. CCR5 and CXCR4 antagonist and agonist binding sites
- Author
-
Paul A. Grieco, Martin Teintze, Royce A. Wilkinson, Edward A. Dratz, and John S. Mills
- Subjects
Agonist ,lcsh:Immunologic diseases. Allergy ,CXCR4 antagonist ,medicine.drug_class ,Biology ,Small molecule ,Infectious Diseases ,Protein structure ,Biochemistry ,Rhodopsin ,Virology ,Immunology ,medicine ,biology.protein ,Oral Presentation ,Binding site ,Receptor ,lcsh:RC581-607 ,G protein-coupled receptor - Abstract
Several small molecule antagonists of the HIV co-receptors CCR5 and CXCR4 are being developed as HIV entry inhibitors, but side effects have been observed in clinical trials that are likely due to agonist activity and/or cross-reactivity with closely related receptors. In order to develop high resolution maps of the binding sites for these antagonists that can be exploited to improve their activity and specificity, we are synthesizing derivatives of CCR5 and CXCR4 antagonists which contain photo-crosslinking groups at a variety positions in the molecules and that retain high affinity and activity against the receptors. Derivatives of two CCR5 antagonists and one CXCR4 antagonist have been crosslinked to their receptors on cells, and the interaction sites are being mapped by mass spectrometry. Techniques for purification, crosslinking, CNBr and/or trypsin digestion, and LC/MS/MS mass spectrometry of highly hydrophobic peptides initially developed using the rhodopsin GPCR system have been applied with some success to CCR5 and CXCR4, although these represent particular challenges due to the relative paucity of Met residue cleavage sites compared to rhodopsin and resistance to tryptic digestion. Nevertheless, peptides covering most of the extracellular and transmembrane regions of CCR5 that could interact with an antagonist can be identified, and affinity purification methods are being developed to isolate CXCR4 peptides crosslinked to derivatives of the antagonist T-140 that should facilitate the identification and localization of the crosslinking sites within the proteins.
- Published
- 2006
27. The history and market impact of CRISPR RNA-guided nucleases
- Author
-
Gary Bloomer, Royce A. Wilkinson, Blake Wiedenheft, and Paul B. G. van Erp
- Subjects
CRISPR-Associated Proteins ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Plasmid ,Virology ,CRISPR ,Clustered Regularly Interspaced Short Palindromic Repeats ,Endodeoxyribonucleases ,030304 developmental biology ,Genetics ,0303 health sciences ,biology ,RNA ,biology.organism_classification ,Restriction enzyme ,chemistry ,biology.protein ,CRISPR-Cas Systems ,Genetic Engineering ,030217 neurology & neurosurgery ,Bacteria ,DNA ,Archaea - Abstract
The interface between viruses and their hosts’ are hot spots for biological and biotechnological innovation. Bacteria use restriction endonucleases to destroy invading DNA, and industry has exploited these enzymes for molecular cut-and-paste reactions that are central to many recombinant DNA technologies. Today, another class of nucleases central to adaptive immune systems that protect bacteria and archaea from invading viruses and plasmids are blazing a similar path from basic science to profound biomedical and industrial applications.
- Full Text
- View/download PDF
28. Antibacterial activity of THAM Trisphenylguanide against methicillin-resistant Staphylococcus aureus.
- Author
-
Alan J Weaver, Joyce B Shepard, Royce A Wilkinson, Robert L Watkins, Sarah K Walton, Amanda R Radke, Thomas J Wright, Milat B Awel, Catherine Cooper, Elizabeth Erikson, Mohamed E Labib, Jovanka M Voyich, and Martin Teintze
- Subjects
Medicine ,Science - Abstract
This study investigated the potential antibacterial activity of three series of compounds synthesized from 12 linear and branched polyamines with 2-8 amino groups, which were substituted to produce the corresponding guanides, biguanides, or phenylguanides, against Acinetobacter baumannii, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. Antibacterial activity was measured for each compound by determining the minimum inhibitory concentration against the bacteria, and the toxicity towards mammalian cells was determined. The most effective compound, THAM trisphenylguanide, was studied in time-to-kill and cytoplasmic leakage assays against methicillin-resistant Staphylococcus aureus (MRSA, USA300) in comparison to chlorhexidine. Preliminary toxicity and MRSA challenge studies in mice were also conducted on this compound. THAM trisphenylguanide showed significant antibacterial activity (MIC ∼1 mg/L) and selectivity against MRSA relative to all the other bacteria examined. In time-to-kill assays it showed increased antimicrobial activity against MRSA versus chlorhexidine. It induced leakage of cytoplasmic content at concentrations that did not reduce cell viability, suggesting the mechanism of action may involve membrane disruption. Using an intraperitoneal mouse model of invasive MRSA disease, THAM trisphenylguanide reduced bacterial burden locally and in deeper tissues. This study has identified a novel guanide compound with selective microbicidal activity against Staphylococcus aureus, including a methicillin-resistant (MRSA) strain.
- Published
- 2014
- Full Text
- View/download PDF
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