13 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
- Author
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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
- Author
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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.
- Published
- 2020
- Full Text
- View/download PDF
8. A PAX5–OCT4–PRDM1 developmental switch specifies human primordial germ cells
- Author
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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
- Author
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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
- Subjects
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
- Subjects
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. 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
- Subjects
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
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