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1.
Mol Biol Evol ; 37(4): 1133-1147, 2020 04 01.
Article in English | MEDLINE | ID: mdl-31873734

ABSTRACT

Evolutionary trajectories are deemed largely irreversible. In a newly diverged protein, reversion of mutations that led to the functional switch typically results in loss of both the new and the ancestral functions. Nonetheless, evolutionary transitions where reversions are viable have also been described. The structural and mechanistic causes of reversion compatibility versus incompatibility therefore remain unclear. We examined two laboratory evolution trajectories of mammalian paraoxonase-1, a lactonase with promiscuous organophosphate hydrolase (OPH) activity. Both trajectories began with the same active-site mutant, His115Trp, which lost the native lactonase activity and acquired higher OPH activity. A neo-functionalization trajectory amplified the promiscuous OPH activity, whereas the re-functionalization trajectory restored the native activity, thus generating a new lactonase that lacks His115. The His115 revertants of these trajectories indicated opposite trends. Revertants of the neo-functionalization trajectory lost both the evolved OPH and the original lactonase activity. Revertants of the trajectory that restored the original lactonase function were, however, fully active. Crystal structures and molecular simulations show that in the newly diverged OPH, the reverted His115 and other catalytic residues are displaced, thus causing loss of both the original and the new activity. In contrast, in the re-functionalization trajectory, reversion compatibility of the original lactonase activity derives from mechanistic versatility whereby multiple residues can fulfill the same task. This versatility enables unique sequence-reversible compositions that are inaccessible when the active site was repurposed toward a new function.


Subject(s)
Aryldialkylphosphatase/genetics , Directed Molecular Evolution , Aryldialkylphosphatase/metabolism , Epistasis, Genetic , Evolution, Molecular , Humans , Phosphoric Monoester Hydrolases/metabolism
2.
J Am Chem Soc ; 139(3): 1155-1167, 2017 01 25.
Article in English | MEDLINE | ID: mdl-28026940

ABSTRACT

Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed ß-propeller with a flexible loop (residues 70-81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1's lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1's lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1's active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar "gating loop" or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates.


Subject(s)
Aryldialkylphosphatase/metabolism , Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/genetics , Binding Sites , Biocatalysis , Humans , Hydrolysis , Hydrophobic and Hydrophilic Interactions , Lactones/chemistry , Lactones/metabolism , Molecular Dynamics Simulation , Mutation , Paraoxon/chemistry , Paraoxon/metabolism , Protein Conformation
3.
Protein Sci ; 33(1): e4824, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37945533

ABSTRACT

The atomic-resolution structural information that X-ray crystallography can provide on the binding interface between a Fab and its cognate antigen is highly valuable for understanding the mechanism of interaction. However, many Fab:antigen complexes are recalcitrant to crystallization, making the endeavor a considerable effort with no guarantee of success. Consequently, there have been significant steps taken to increase the likelihood of Fab:antigen complex crystallization by altering the Fab framework. In this investigation, we applied the surface entropy reduction strategy coupled with phage-display technology to identify a set of surface substitutions that improve the propensity of a human Fab framework to crystallize. In addition, we showed that combining these surface substitutions with previously reported Crystal Kappa and elbow substitutions results in an extraordinary improvement in Fab and Fab:antigen complex crystallizability, revealing a strong synergistic relationship between these sets of substitutions. Through comprehensive Fab and Fab:antigen complex crystallization screenings followed by structure determination and analysis, we defined the roles that each of these substitutions play in facilitating crystallization and how they complement each other in the process.


Subject(s)
Antigen-Antibody Complex , Immunoglobulin Fab Fragments , Humans , Crystallization/methods , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/chemistry , Antigen-Antibody Complex/chemistry , Antigens/chemistry , Crystallography, X-Ray , Protein Conformation
4.
Nat Chem Biol ; 7(2): 120-5, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21217689

ABSTRACT

Organophosphate nerve agents are extremely lethal compounds. Rapid in vivo organophosphate clearance requires bioscavenging enzymes with catalytic efficiencies of >10(7) (M(-1) min(-1)). Although serum paraoxonase (PON1) is a leading candidate for such a treatment, it hydrolyzes the toxic S(p) isomers of G-agents with very slow rates. We improved PON1's catalytic efficiency by combining random and targeted mutagenesis with high-throughput screening using fluorogenic analogs in emulsion compartments. We thereby enhanced PON1's activity toward the coumarin analog of S(p)-cyclosarin by ∼10(5)-fold. We also developed a direct screen for protection of acetylcholinesterase from inactivation by nerve agents and used it to isolate variants that degrade the toxic isomer of the coumarin analog and cyclosarin itself with k(cat)/K(M) ∼ 10(7) M(-1) min(-1). We then demonstrated the in vivo prophylactic activity of an evolved variant. These evolved variants and the newly developed screens provide the basis for engineering PON1 for prophylaxis against other G-type agents.


Subject(s)
Chemical Warfare Agents/poisoning , Directed Molecular Evolution , Hydrolases/metabolism , Poisoning/prevention & control , Acetylcholinesterase/metabolism , Aryldialkylphosphatase/metabolism , Biocatalysis , Emulsions , Hydrolases/chemistry , Hydrolysis
5.
Nanomedicine ; 8(5): 569-79, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22024194

ABSTRACT

Magnetoacoustic detection is a new method for the noninvasive, early detection of cancer. It uses specific superparamagnetic nanoparticles (NPs) that bind to tumor sites together with magnetic excitation and acoustic detection of the tumor-NPs complex. This work tests the feasibility of such method theoretically and experimentally. An extensive analytic model has been developed that shows an ability to detect small tumors, a few centimeters deep inside the tissue. A series of experiments were conducted to validate the theoretical model. The performance of specially designed solenoids was measured, and the detection of the tumor presence in phantom was demonstrated. Experimental results agree well with the theoretical calculations, providing preliminary proof of concept. We demonstrate the ability to detect a 5-mm diameter spherical tumor located 3 cm deep. Instrumentation and measurements are inexpensive and accurate. The accuracy, speed, and costs of this method show the potential for early detection of cancer. FROM THE CLINICAL EDITOR: A sensitive and cost effective magentoacoustic tumor detection method is presented in this paper using superparamagnetic nanoparticles. The method is demonstrated in a phantom by detecting a 5-mm diameter spherical tumor located 3 cm deep.


Subject(s)
Antibodies , Magnetite Nanoparticles , Neoplasms/diagnosis , Antibodies/chemistry , Humans , Magnetite Nanoparticles/chemistry , Sound
6.
Opt Express ; 19(21): 19822-35, 2011 Oct 10.
Article in English | MEDLINE | ID: mdl-21996990

ABSTRACT

Layered peptide array (LPA) system enables multiplex screening of biomarkers [1-3]. One of the main problems of the LPA system is the screening of the layered-membranes stack. Nowadays, each membrane is imaged separately using conventional fluorescent imaging. This process is time consuming and requires extensive manual interaction. This paper describes a general solution for optical imaging of a layered grid medium using photogrammetric methods. The suggested method enables visualization of the LPA membranes stack by using only two images of the stack. This study is a proof of concept of the suggested solution using MATLAB simulation and phantom experiments.


Subject(s)
Agar/chemistry , Peptides/chemistry , Algorithms , Calibration , Computer Simulation , Equipment Design , Fluorescent Dyes/pharmacology , Gels , Imaging, Three-Dimensional , Microscopy, Fluorescence/methods , Optics and Photonics/methods , Phantoms, Imaging , Reproducibility of Results , Software
7.
Med Phys ; 37(1): 211-6, 2010 Jan.
Article in English | MEDLINE | ID: mdl-20175483

ABSTRACT

PURPOSE: This article deals with the development of a minimal-invasive, infrared (IR) (8-12 microm spectral range) imaging technique that would improve upon current methods by using superparamagnetic nanostructured core/shell particles for imaging as well as for therapy. This technique may function as a diagnostic tool, thanks to the ability of specific bioconjugation of these nanoparticles to a tumor's outer surface. Hence, by applying an alternating magnetic field, the authors could cause a selective elevation of temperature of the nanoparticles for +1 - +5 degrees C, enabling tumor's imaging. Further elevation of the temperature over +10 degrees C will cause a necrotic effect, leading to localized irreversible damage to the cancerous site without harming the surrounding tissues. This technique may also serve as a targeted therapeutic tool under thermal feedback control. METHODS: Under alternative magnetic field, these biocompatible nanoparticles can generate heat, which propagates along the tissue (by thermal conduction), reaching the tissue's surface. Surface temperature distribution can be acquired by an IR camera and analyzed to retrieve nanoparticles' temperature and location within the tissue. An analytical-based steady-state solution for the thermal inverse problem was developed, considering an embedded point heat source. Based on this solution, the authors developed an algorithm that generates solutions for the corresponding forward problem, and based on discovered relations between the problem's characteristic, can derive the depth and temperature of the embedded heat source from the surface temperature profile, derived from the thermal image. RESULTS: The algorithm was able to compute the heat source depth and power (proportional to its temperature) in two phases. Assuming that the surface temperature profile can be fitted to a Lorentzian curve, the first phase computing the source depth was based on a linear relation between the depth and the FWHM value of the surface temperature profile, which is independent of the source power. This relation varies between different tissues and surface conditions. The second phase computing the power (Q) was based on an exponential relation between the area (A) curve of the surface temperature profile and power (Q), dependent on the depth computed in the first phase. The simulation results show that given the tissue thermal properties, the surface conductance, and the ambient conditions, an inverse solution can be applied retrieving the depth and temperature of a point heat source from a 2D thermal image. CONCLUSIONS: The predicted depth and heat source power were compared to the actual parameters (which were derived). Differences between the real and estimated values may occur primarily in computing the forward solution, which was used for the estimation itself. The fact that the computation is carried out discretely and the spatial resolution in the radial direction are influencing factors. To improve and eliminate these factors, the resolution may be increased or suitable interpolation and/or smoothing may be applied. Applying this algorithm on a spherical heat source volume may be feasible. A solution for the forward problem was established, yet incorporation of the source radius has to be further examined.


Subject(s)
Algorithms , Image Interpretation, Computer-Assisted/methods , Models, Biological , Neoplasms/diagnosis , Neoplasms/physiopathology , Thermography/methods , Computer Simulation , Humans , Infrared Rays , Reproducibility of Results , Sensitivity and Specificity
8.
Appl Opt ; 49(25): 4700-9, 2010 Sep 01.
Article in English | MEDLINE | ID: mdl-20820210

ABSTRACT

There has been very little work done in the past to extend the wavelength range of fiber image bundles to the IR range. This is due, in part, to the lack of IR transmissive fibers with optical and mechanical properties analogous to the oxide glass fibers currently employed in the visible fiber bundles. Our research is aimed at developing high-resolution hollow-core coherent IR fiber bundles for transendoscopic infrared imaging. We employ the hollow glass waveguide (HGW) technology that was used successfully to make single-HGWs with Ag/AgI thin film coatings to form coherent bundles for IR imaging. We examine the possibility of developing endoscopic systems to capture thermal images using hollow waveguide fiber bundles adjusted to the 8-10?mum spectral range and investigate the applicability of such systems. We carried out a series of measurements in order to characterize the optical properties of the fiber bundles. These included the attenuation, resolution, and temperature response. We developed theoretical models and simulation tools that calculate the light propagation through HGW bundles, and which can be used to calculate the optical properties of the fiber bundles. Finally, the HGW fiber bundles were used to transmit thermal images of various heated objects; the results were compared with simulation results. The experimental results are encouraging, show an improvement in the resolution and thermal response of the HGW fiber bundles, and are consistent with the theoretical results. Nonetheless, additional improvements in the attenuation of the bundles are required in order to be able to use this technology for medical applications.


Subject(s)
Diagnostic Imaging/methods , Endoscopy/methods , Infrared Rays , Optical Fibers , Computer Simulation , Humans , Temperature
9.
Nanomedicine ; 6(6): 786-96, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20620238

ABSTRACT

This work describes the utilization of tumor-specific magnetic nanoparticles together with an alternating magnetic field as a means to thermally mark a tumor so as to detect it using a thermal imaging system. Experiments were conducted using an in vitro tissue model, an inductive heating system, and an infrared camera. The thermal images, recorded by the infrared camera during the experiments, were analyzed using an algorithm that was developed as part of this work. The results show that small tumor phantoms (diameter of 0.5 mm) that were embedded under the surface of the tissue phantom (up to 14 mm below the surface) can be detected and located, indicating that the proposed method could potentially offer considerable advantages over conventional thermography and other methods for cancer early detection. Nevertheless, several issues should be clarified in future studies before the method can be offered for clinical use.


Subject(s)
Magnetics , Nanoparticles , Neoplasms/diagnosis , Thermography , Algorithms , Humans , Models, Theoretical , Reproducibility of Results
10.
Proteins ; 77 Suppl 9: 50-65, 2009.
Article in English | MEDLINE | ID: mdl-19774550

ABSTRACT

The biennial CASP experiment is a crucial way to evaluate, in an unbiased way, the progress in predicting novel 3D protein structures. In this article, we assess the quality of prediction of template free models, that is, ab initio prediction of 3D structures of proteins based solely on the amino acid sequences, that is, proteins that did not have significant sequence identity to any protein in the Protein Data Bank. There were 13 targets in this category and 102 groups submitted predictions. Analysis was based on the GDT_TS analysis, which has been used in previous CASP experiments, together with a newly developed method, the OK_Rank, as well as by visual inspection. There is no doubt that in recent years many obstacles have been removed on the long and elusive way to deciphering the protein-folding problem. Out of the 13 targets, six were predicted well by a number of groups. On the other hand, it must be stressed that for four targets, none of the models were judged to be satisfactory. Thus, for template free model prediction, as evaluated in this CASP, successes have been achieved for most targets; however, a great deal of research is still required, both in improving the existing methods and in development of new approaches.


Subject(s)
Computational Biology/methods , Models, Molecular , Proteins/chemistry , Sequence Analysis, Protein/methods , Amino Acid Sequence , Databases, Protein , Protein Folding , Protein Structure, Secondary
11.
J Mol Biol ; 431(2): 336-350, 2019 01 18.
Article in English | MEDLINE | ID: mdl-30471255

ABSTRACT

Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.


Subject(s)
Allosteric Regulation/physiology , Protein Binding/physiology , Proto-Oncogene Proteins c-fyn/metabolism , src Homology Domains/physiology , Amino Acid Sequence , Humans , Hydrophobic and Hydrophilic Interactions , Ligands , Molecular Dynamics Simulation , Protein Conformation
12.
Curr Opin Struct Biol ; 45: 25-35, 2017 08.
Article in English | MEDLINE | ID: mdl-27866084

ABSTRACT

Recent studies on genome sequencing and genetic screens with RNAi and CRISPR technology have revolutionized our understanding of aberrant signaling networks in human diseases. A strategy combining both genetic and protein-based technologies should be at the heart of modern drug-development efforts, particularly in the era of precision medicine. Thus, there is an urgent need for efficient platforms to develop probes that can modulate protein function in cells to validate drug targets and to develop therapeutic leads. Advanced protein engineering has enabled the rapid production of monoclonal antibodies and small protein scaffold affinity reagents for diverse protein targets. Here, we review the most recent progress on engineering natural protein-protein interactions for modulation of cell signaling.


Subject(s)
Protein Engineering/methods , Protein Interaction Maps , Signal Transduction/genetics , Animals , Humans , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin/metabolism
13.
J Mol Biol ; 427(6 Pt B): 1359-1374, 2015 Mar 27.
Article in English | MEDLINE | ID: mdl-25644661

ABSTRACT

Despite the abundance of membrane-associated enzymes, the mechanism by which membrane binding stabilizes these enzymes and stimulates their catalysis remains largely unknown. Serum paraoxonase-1 (PON1) is a lipophilic lactonase whose stability and enzymatic activity are dramatically stimulated when associated with high-density lipoprotein (HDL) particles. Our mutational and structural analyses, combined with empirical valence bond simulations, reveal a network of hydrogen bonds that connect HDL binding residues with Asn168--a key catalytic residue residing >15Å from the HDL contacting interface. This network ensures precise alignment of N168, which, in turn, ligates PON1's catalytic calcium and aligns the lactone substrate for catalysis. HDL binding restrains the overall motion of the active site and particularly of N168, thus reducing the catalytic activation energy barrier. We demonstrate herein that disturbance of this network, even at its most far-reaching periphery, undermines PON1's activity. Membrane binding thus immobilizes long-range interactions via second- and third-shell residues that reduce the active site's floppiness and pre-organize the catalytic residues. Although this network is critical for efficient catalysis, as demonstrated here, unraveling these long-rage interaction networks is challenging, let alone their implementation in artificial enzyme design.


Subject(s)
Aryldialkylphosphatase/chemistry , Aryldialkylphosphatase/metabolism , Lipoproteins, HDL/metabolism , Aryldialkylphosphatase/genetics , Calcium/metabolism , Carboxylic Ester Hydrolases/chemistry , Carboxylic Ester Hydrolases/metabolism , Catalysis , Catalytic Domain , Computer Simulation , Crystallography, X-Ray , Humans , Hydrogen Bonding , Kinetics , Lipoproteins, HDL/chemistry , Models, Molecular , Mutation/genetics , Protein Conformation
14.
J Mol Biol ; 425(6): 1028-38, 2013 Mar 25.
Article in English | MEDLINE | ID: mdl-23318950

ABSTRACT

Although largely deemed as structurally conserved, catalytic metal ion sites can rearrange, thereby contributing to enzyme evolvability. Here, we show that in paraoxonase-1, a lipo-lactonase, catalytic promiscuity and divergence into an organophosphate hydrolase are correlated with an alternative mode of the catalytic Ca(2+). We describe the crystal structures of active-site mutants bearing mutations at position 115. The histidine at this position acts as a base to activate the lactone-hydrolyzing water molecule. Mutations to Trp or Gln indeed diminish paraoxonase-1's lactonase activity; however, the promiscuous organophosphate hydrolase activity is enhanced. The structures reveal a 1.8-Å upward displacement towards the enzyme's surface of the catalytic Ca(2+) in the His115 mutants and configurational changes in the ligating side chains and water molecules, relative to the wild-type enzyme. Biochemical analysis and molecular dynamics simulations suggest that this alternative, upward metal mode mediates the promiscuous hydrolysis of organophosphates. The upward Ca(2+) mode observed in the His115 mutants also appears to mediate the wild type's paraoxonase activity. However, whereas the upward mode dominates in the Trp115 mutant, it is scarcely populated in wild type. Thus, the plasticity of active-site metal ions may permit alternative, latent, promiscuous activities and also provide the basis for the divergence of new enzymatic functions.


Subject(s)
Aryldialkylphosphatase/chemistry , Metalloproteins/chemistry , Aryldialkylphosphatase/genetics , Aryldialkylphosphatase/metabolism , Binding Sites , Calcium/metabolism , Catalysis , Catalytic Domain , Crystallography, X-Ray , Histidine/genetics , Hydrolysis , Lactones/chemistry , Lactones/metabolism , Metalloproteins/genetics , Metalloproteins/metabolism , Models, Molecular , Mutagenesis, Site-Directed , Protein Conformation
15.
J Mol Biol ; 418(3-4): 181-96, 2012 May 04.
Article in English | MEDLINE | ID: mdl-22387469

ABSTRACT

The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential.


Subject(s)
Aryldialkylphosphatase/chemistry , Animals , Aryldialkylphosphatase/blood , Binding Sites , Catalysis , Catalytic Domain , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Models, Molecular , Protein Conformation , Rabbits , Recombinant Proteins/chemistry , Substrate Specificity
16.
Chem Biol ; 19(4): 456-66, 2012 Apr 20.
Article in English | MEDLINE | ID: mdl-22520752

ABSTRACT

A preferred strategy for preventing nerve agents intoxication is catalytic scavenging by enzymes that hydrolyze them before they reach their targets. Using directed evolution, we simultaneously enhanced the activity of a previously described serum paraoxonase 1 (PON1) variant for hydrolysis of the toxic S(P) isomers of the most threatening G-type nerve agents. The evolved variants show ≤340-fold increased rates and catalytic efficiencies of 0.2-5 × 10(7) M(-1) min(-1). Our selection for prevention of acetylcholinesterase inhibition also resulted in the complete reversion of PON1's stereospecificity, from an enantiomeric ratio (E) < 6.3 × 10(-4) in favor of the R(P) isomer of a cyclosarin analog in wild-type PON1, to E > 2,500 for the S(P) isomer in an evolved variant. Given their ability to hydrolyze G-agents, these evolved variants may serve as broad-range G-agent prophylactics.


Subject(s)
Aryldialkylphosphatase/metabolism , Chemical Warfare Agents/metabolism , Aryldialkylphosphatase/genetics , Biocatalysis , Biotransformation , Chemical Warfare Agents/toxicity , Humans , Hydrolysis , Mutation , Stereoisomerism , Substrate Specificity
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