26 results found
Macé K, Vadakkepat AK, Redzej A, et al., 2022, Cryo-EM structure of a type IV secretion system, Nature, Vol: 607, ISSN: 0028-0836
Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.
Low WW, Wong J, Beltran L, et al., 2022, Mating pair stabilization mediates bacterial conjugation species specificity, Nature Microbiology, Vol: 7, Pages: 1016-1027, ISSN: 2058-5276
Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating thespread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane(OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNAtransfer. We show that TraN from the plasmids pKpQIL (Klebsiella pneumoniae), R100-1 (Shigella flexneri) and pSLT (SalmonellaTyphimurium), and the prototypical F plasmid (Escherichia coli) interact with OmpK36, OmpW and OmpA, respectively.Cryo-EM analysis revealed that TraN pKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN intoa monomer of the OmpK36 trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified afourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classifica-tion scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ(OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect thedistribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pairstabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emergingresistance plasmids in high-risk bacterial pathogens.
Lockwood DC, Amin H, Costa TRD, et al., 2022, The Legionella pneumophila Dot/Icm type IV secretion system and its effectors, Microbiology, Vol: 168, ISSN: 1350-0872
To prevail in the interaction with eukaryotic hosts, many bacterial pathogens use protein secretion systems to release virulence factors at the host–pathogen interface and/or deliver them directly into host cells. An outstanding example of the complexity and sophistication of secretion systems and the diversity of their protein substrates, effectors, is the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) Type IVB secretion system (T4BSS) of Legionella pneumophila and related species. Legionella species are facultative intracellular pathogens of environmental protozoa and opportunistic human respiratory pathogens. The Dot/Icm T4BSS translocates an exceptionally large number of effectors, more than 300 per L. pneumophila strain, and is essential for evasion of phagolysosomal degradation and exploitation of protozoa and human macrophages as replicative niches. Recent technological advancements in the imaging of large protein complexes have provided new insight into the architecture of the T4BSS and allowed us to propose models for the transport mechanism. At the same time, significant progress has been made in assigning functions to about a third of L. pneumophila effectors, discovering unprecedented new enzymatic activities and concepts of host subversion. In this review, we describe the current knowledge of the workings of the Dot/Icm T4BSS machinery and provide an overview of the activities and functions of the to-date characterized effectors in the interaction of L. pneumophila with host cells.
Kreutzberger MAB, Sobe RC, Sauder AB, et al., 2022, Flagellin outer domain dimerization modulates motility in pathogenic and soil bacteria from viscous environments., Nature Communications, Vol: 13, Pages: 1-13, ISSN: 2041-1723
Flagellar filaments function as the propellers of the bacterial flagellum and their supercoiling is key to motility. The outer domains on the surface of the filament are non-critical for motility in many bacteria and their structures and functions are not conserved. Here, we show the atomic cryo-electron microscopy structures for flagellar filaments from enterohemorrhagic Escherichia coli O157:H7, enteropathogenic E. coli O127:H6, Achromobacter, and Sinorhizobium meliloti, where the outer domains dimerize or tetramerize to form either a sheath or a screw-like surface. These dimers are formed by 180° rotations of half of the outer domains. The outer domain sheath (ODS) plays a role in bacterial motility by stabilizing an intermediate waveform and prolonging the tumbling of E. coli cells. Bacteria with these ODS and screw-like flagellar filaments are commonly found in soil and human intestinal environments of relatively high viscosity suggesting a role for the dimerization in these environments.
Amin H, Ilangovan A, Costa TRD, 2022, Publisher Correction: Architecture of the outer-membrane core complex from a conjugative type IV secretion system., Nature Communications, Vol: 13, Pages: 1-1, ISSN: 2041-1723
Amin H, Ilangovan A, Costa TRD, 2021, Architecture of the outer-membrane core complex from a conjugative type IV secretion system., Nat Commun, Vol: 12
Conjugation is one of the most important processes that bacteria utilize to spread antibiotic resistance genes among bacterial populations. Interbacterial DNA transfer requires a large double membrane-spanning nanomachine called the type 4 secretion system (T4SS) made up of the inner-membrane complex (IMC), the outer-membrane core complex (OMCC) and the conjugative pilus. The iconic F plasmid-encoded T4SS has been central in understanding conjugation for several decades, however atomic details of its structure are not known. Here, we report the structure of a complete conjugative OMCC encoded by the pED208 plasmid from E. coli, solved by cryo-electron microscopy at 3.3 Å resolution. This 2.1 MDa complex has a unique arrangement with two radial concentric rings, each having a different symmetry eventually contributing to remarkable differences in protein stoichiometry and flexibility in comparison to other OMCCs. Our structure suggests that F-OMCC is a highly dynamic complex, with implications for pilus extension and retraction during conjugation.
Howard SA, Furniss RCD, Bonini D, et al., 2021, The breadth and molecular basis of Hcp-driven type six secretion system (T6SS) effector delivery, mBio, Vol: 12, Pages: 1-19, ISSN: 2150-7511
The type VI secretion system (T6SS) is a bacterial nanoscale weapon that delivers toxins into prey ranging from bacteria and fungi to animal hosts. The cytosolic contractile sheath of the system wraps around stacked hexameric rings of Hcp proteins, which form an inner tube. At the tip of this tube is a puncturing device comprising a trimeric VgrG topped by a monomeric PAAR protein. The number of toxins a single system delivers per firing event remains unknown, since effectors can be loaded on diverse sites of the T6SS apparatus, notably the inner tube and the puncturing device. Each VgrG or PAAR can bind one effector, and additional effector cargoes can be carried in the Hcp ring lumen. While many VgrG- and PAAR-bound toxins have been characterized, to date, very few Hcp-bound effectors are known. Here, we used 3 known Pseudomonas aeruginosa Hcp proteins (Hcp1 to -3), each of which associates with one of the three T6SSs in this organism (H1-T6SS, H2-T6SS, and H3-T6SS), to perform in vivo pulldown assays. We confirmed the known interactions of Hcp1 with Tse1 to -4, further copurified a Hcp1-Tse4 complex, and identified potential novel Hcp1-bound effectors. Moreover, we demonstrated that Hcp2 and Hcp3 can shuttle T6SS cargoes toxic to Escherichia coli. Finally, we used a Tse1-Bla chimera to probe the loading strategy for Hcp passengers and found that while large effectors can be loaded onto Hcp, the formed complex jams the system, abrogating T6SS function.
Zheng W, Peña A, Ilangovan A, et al., 2021, Cryoelectron-microscopy structure of the enteropathogenic Escherichia coli type III secretion system EspA filament., Proceedings of the National Academy of Sciences of USA, Vol: 118, ISSN: 0027-8424
Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) utilize a macromolecular type III secretion system (T3SS) to inject effector proteins into eukaryotic cells. This apparatus spans the inner and outer bacterial membranes and includes a helical needle protruding into the extracellular space. Thus far observed only in EPEC and EHEC and not found in other pathogenic Gram-negative bacteria that have a T3SS is an additional helical filament made by the EspA protein that forms a long extension to the needle, mediating both attachment to eukaryotic cells and transport of effector proteins through the intestinal mucus layer. Here, we present the structure of the EspA filament from EPEC at 3.4 Å resolution. The structure reveals that the EspA filament is a right-handed 1-start helical assembly with a conserved lumen architecture with respect to the needle to ensure the seamless transport of unfolded cargos en route to the target cell. This functional conservation is despite the fact that there is little apparent overall conservation at the level of sequence or structure with the needle. We also unveil the molecular details of the immunodominant EspA epitope that can now be exploited for the rational design of epitope display systems.
Costa TRD, Harb L, Khara P, et al., 2020, Type IV secretion systems: Advances in structure, function, and activation., Molecular Microbiology, ISSN: 0950-382X
Bacterial type IV secretion systems (T4SSs) are a functionally diverse translocation superfamily. They consist mainly of two large subfamilies: (i) conjugation systems that mediate interbacterial DNA transfer and (ii) effector translocators that deliver effector macromolecules into prokaryotic or eukaryotic cells. A few other T4SSs export DNA or proteins to the milieu, or import exogenous DNA. The T4SSs are defined by 6 or 12 conserved "core" subunits that respectively elaborate "minimized" systems in Gram-positive or -negative bacteria. However, many "expanded" T4SSs are built from "core" subunits plus numerous others that are system-specific, which presumptively broadens functional capabilities. Recently, there has been exciting progress in defining T4SS assembly pathways and architectures using a combination of fluorescence and cryoelectron microscopy. This review will highlight advances in our knowledge of structure-function relationships for model Gram-negative bacterial T4SSs, including "minimized" systems resembling the Agrobacterium tumefaciens VirB/VirD4 T4SS and "expanded" systems represented by the Helicobacter pylori Cag, Legionella pneumophila Dot/Icm, and F plasmid-encoded Tra T4SSs. Detailed studies of these model systems are generating new insights, some at atomic resolution, to long-standing questions concerning mechanisms of substrate recruitment, T4SS channel architecture, conjugative pilus assembly, and machine adaptations contributing to T4SS functional versatility.
Williams AH, Redzej A, Rolhion N, et al., 2019, The cryo-electron microscopy supramolecular structure of the bacterial stressosome unveils its mechanism of activation, Nature Communications, Vol: 10, ISSN: 2041-1723
How the stressosome, the epicenter of the stress response in bacteria, transmits stress signals from the environment has remained elusive. The stressosome consists of multiple copies of three proteins RsbR, RsbS and RsbT, a kinase that is important for its activation. Using cryo-electron microscopy, we determined the atomic organization of the Listeria monocytogenes stressosome at 3.38 Å resolution. RsbR and RsbS are organized in a 60-protomers truncated icosahedron. A key phosphorylation site on RsbR (T209) is partially hidden by an RsbR flexible loop, whose "open" or "closed" position could modulate stressosome activity. Interaction between three glutamic acids in the N-terminal domain of RsbR and the membrane-bound mini-protein Prli42 is essential for Listeria survival to stress. Together, our data provide the atomic model of the stressosome core and highlight a loop important for stressosome activation, paving the way towards elucidating the mechanism of signal transduction by the stressosome in bacteria.
Costa TRD, Francis MK, Farag SI, et al., 2019, Measurement of Yersinia Translocon Pore Formation in Erythrocytes, Methods in Molecular Biology, Publisher: Springer New York, Pages: 211-229, ISBN: 9781493995400
Sgro GG, Costa TRD, Cenens W, et al., 2018, Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri, Nature Microbiology, Vol: 3, Pages: 1429-1440, ISSN: 2058-5276
Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein–protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.
S Sgro G, Costa TRD, 2018, Cryo-EM grid preparation of membrane protein samples for single particle analysis, Frontiers in Molecular Biosciences, Vol: 5, ISSN: 2296-889X
Recent advances in cryo-electron microscopy (cryo-EM) have made it possible to solve structures of biological macromolecules at near atomic resolution. Development of more stable microscopes, improved direct electron detectors and faster software for image processing has enabled structural solution of not only large macromolecular (megadalton range) complexes but also small (~60 kDa) proteins. As a result of the widespread use of the technique, we have also witnessed new developments of techniques for cryo-EM grid preparation of membrane protein samples. This includes new types of solubilization strategies that better stabilize these protein complexes and the development of new grid supports with proven efficacy in reducing the motion of the molecules during electron beam exposure. Here, we discuss the practicalities and recent challenges of membrane protein sample preparation and vitrification, as well as grid support and foil treatment in the context of the structure determination of protein complexes by single particle cryo-EM.
Gurung JM, Amer AAA, Francis MK, et al., 2018, Heterologous complementation studies with the YscX and YscY protein families reveals a specificity for Yersinia pseudotuberculosis Type III secretion, Frontiers in Cellular and Infection Microbiology, Vol: 8, Pages: 1-1, ISSN: 2235-2988
Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenic Yersinia species, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither an yscX nor yscY null mutant of Yersinia is capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems of Pseudomonas aeruginosa (PscX and PscY), Aeromonas species (AscX and AscY), Vibrio species (VscX and VscY), and Photorhabdus luminescens (SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the native yscX in the near full-length yscX deletion and native yscY in the near full-length yscY deletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical for Yersinia type III secretion.
Hospenthal MK, Zyla D, Costa TRD, et al., 2017, The Cryoelectron Microscopy Structure of the Type 1 Chaperone-Usher Pilus Rod, STRUCTURE, Vol: 25, Pages: 1829-+, ISSN: 0969-2126
- Author Web Link
- Citations: 24
Gordon JE, Costa TRD, Patel RS, et al., 2017, Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation, MOLECULAR MICROBIOLOGY, Vol: 105, Pages: 273-293, ISSN: 0950-382X
- Author Web Link
- Citations: 30
Costa TRD, Ignatiou A, Orlova EV, 2017, Structural Analysis of Protein Complexes by Cryo Electron Microscopy., Methods Mol Biol, Vol: 1615, Pages: 377-413
Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.
Hospenthal MK, Costa TRD, Waksman G, 2017, A comprehensive guide to pilus biogenesis in Gram-negative bacteria, NATURE REVIEWS MICROBIOLOGY, Vol: 15, Pages: 365-379, ISSN: 1740-1526
Francis MS, Amer AAA, Milton DL, et al., 2016, Site-Directed Mutagenesis and Its Application in Studying the Interactions of T3S Components., Methods Mol Biol, Vol: 1531, Pages: 11-31
Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections-unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.
Costa TRD, Ilangovan A, Ukleja M, et al., 2016, Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex, CELL, Vol: 166, Pages: 1436-1444.e10, ISSN: 0092-8674
Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F “sex” pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.
Amer AAA, Gurung JM, Costa TRD, et al., 2016, YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis, FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, Vol: 6, ISSN: 2235-2988
Yersiniabacteria target Yop effector toxins to the interior of host immune cells by theYsc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controllingYsc-Yop targeting activity. A+1 frameshift event in the 3-prime end ofyopNcan alsoproduce a singular secreted YopN-TyeA polypeptide that retains some regulatory functioneven though the C-terminal coding sequence of this YopN differs greatly from wild type.Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control.Bacteria producing YopN truncated after residue 278, or with altered sequence betweenresidues 279 and 287, had lost type III secretion control and function. In contrast,YopN variants with manipulated sequence beyond residue 287 maintained full controland function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279Gmutantlost all ability to bind TyeA. The TyeA residue F8was also critical for reciprocal YopNbinding. Thus, we conclude that specific hydrophobic contacts between opposing YopNand TyeA termini establishes a complex needed for regulating Ysc-Yop activity.
Costa TRD, Felisberto-Rodrigues C, Meir A, et al., 2015, Secretion systems in Gram-negative bacteria: structural and mechanistic insights, NATURE REVIEWS MICROBIOLOGY, Vol: 13, Pages: 343-359, ISSN: 1740-1526
- Author Web Link
- Citations: 578
Amer AAA, Costa TRD, Farag SI, et al., 2013, Genetically Engineered Frameshifted YopN-TyeA Chimeras Influence Type III Secretion System Function in Yersinia pseudotuberculosis, PLOS ONE, Vol: 8, ISSN: 1932-6203
Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize theireukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cellcytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known fortheir roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeAheterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybridprotein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for theclosely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis,we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybridby introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeApolypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid.Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficientlysecreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structuresand controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most geneticallyaltered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. Thiscorresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to thesame extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineeredpolypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S controland activity
Costa TRD, Amer AAA, Farag SI, et al., 2013, Type III secretion translocon assemblies that attenuate Yersinia virulence, CELLULAR MICROBIOLOGY, Vol: 15, Pages: 1088-1110, ISSN: 1462-5814
- Author Web Link
- Citations: 14
Costa TRD, Amer AAA, Fallman M, et al., 2012, Coiled-coils in the YopD translocator family: A predicted structure unique to the YopD N-terminus contributes to full virulence of Yersinia pseudotuberculosis, INFECTION GENETICS AND EVOLUTION, Vol: 12, Pages: 1729-1742, ISSN: 1567-1348
- Author Web Link
- Citations: 6
Costa TRD, Edqvist PJ, Broems JE, et al., 2010, YopD Self-assembly and Binding to LcrV Facilitate Type III Secretion Activity by Yersinia pseudotuberculosis, JOURNAL OF BIOLOGICAL CHEMISTRY, Vol: 285, Pages: 25269-25284
- Author Web Link
- Citations: 22
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