scholarly journals A Nanobody Targeting Viral Nonstructural Protein 9 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Replication

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Lizhen Wang ◽  
Lu Zhang ◽  
Baichen Huang ◽  
Kuokuo Li ◽  
Gaopeng Hou ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Respiratory Syndrome Virus ◽  
Dependent Manner ◽  
Swine Industry ◽  
Content Type ◽  
E Coli ◽  
Genotype 2 ◽  
Infected Cells

ABSTRACTPorcine reproductive and respiratory syndrome (PRRS) is of great concern to the swine industry due to pandemic outbreaks of the disease, current ineffective vaccinations, and a lack of efficient antiviral strategies. In our previous study, a PRRSV Nsp9-specific nanobody, Nb6, was successfully isolated, and the intracellularly expressed Nb6 could dramatically inhibit PRRSV replication in MARC-145 cells. However, despite its small size, the application of Nb6 protein in infected cells is greatly limited, as the protein itself cannot enter the cells physically. In this study, atrans-activating transduction (TAT) peptide was fused with Nb6 to promote protein entry into cells. TAT-Nb6 was expressed as an inclusion body inEscherichia coli, and indirect enzyme-linked immunosorbent assays and pulldown assays showed thatE. coli-expressed TAT-Nb6 maintained the binding ability toE. coli-expressed or PRRSV-encoded Nsp9. We demonstrated that TAT delivered Nb6 into MARC-145 cells and porcine alveolar macrophages (PAMs) in a dose- and time-dependent manner, and TAT-Nb6 efficiently inhibited the replication of several PRRSV genotype 2 strains as well as a genotype 1 strain. Using a yeast two-hybrid assay, Nb6 recognition sites were identified in the C-terminal part of Nsp9 and spanned two discontinuous regions (Nsp9aa454–551and Nsp9aa599–646). Taken together, these results suggest that TAT-Nb6 can be developed as an antiviral drug for the inhibition of PRRSV replication and controlling PRRS disease.IMPORTANCEThe pandemic outbreak of PRRS, which is caused by PRRSV, has greatly affected the swine industry. We still lack an efficient vaccine, and it is an immense challenge to control its infection. An intracellularly expressed Nsp9-specific nanobody, Nb6, has been shown to be able to inhibit PRRSV replication in MARC-145 cells. However, its application is limited, because Nb6 cannot physically enter cells. Here, we demonstrated that the cell-penetrating peptide TAT could deliver Nb6 into cultured cells. In addition, TAT-Nb6 fusion protein could suppress the replication of various PRRSV strains in MARC-145 cells and PAMs. These findings may provide a new approach for drug development to control PRRS.

scholarly journals Structural Biology of the Arterivirus nsp11 Endoribonucleases

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Manfeng Zhang ◽  
Xiaorong Li ◽  
Zengqin Deng ◽  
Zhenhang Chen ◽  
Yang Liu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
The United States ◽  
Equine Arteritis Virus ◽  
Respiratory Syndrome Virus ◽  
Human Pathogens ◽  
Content Type ◽  
Terminal Domain ◽  
Insight Into

ABSTRACT Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the United States and almost €1.5 billion in Europe every year. To find a way to combat these viruses, we focused on the essential viral nonstructural protein 11 (nsp11). nsp11 is associated with multiple functions, such as RNA processing and suppression of the infected host innate immunity system. The three structures solved in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

scholarly journals The Inhaled Steroid Ciclesonide Blocks SARS-CoV-2 RNA Replication by Targeting the Viral Replication-Transcription Complex in Cultured Cells

2020 ◽  
Vol 95 (1) ◽  
Author(s):  
Shutoku Matsuyama ◽  
Miyuki Kawase ◽  
Naganori Nao ◽  
Kazuya Shirato ◽  
Makoto Ujike ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cultured Cells ◽  
Inflammatory Responses ◽  
Hepatitis Virus ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
Rna Replication ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Inhaled Corticosteroid

ABSTRACT Here, we screened steroid compounds to obtain a drug expected to block host inflammatory responses and Middle East respiratory syndrome coronavirus (MERS-CoV) replication. Ciclesonide, an inhaled corticosteroid, suppressed the replication of MERS-CoV and other coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), in cultured cells. The 90% effective concentration (EC90) of ciclesonide for SARS-CoV-2 in differentiated human bronchial tracheal epithelial cells was 0.55 μM. Eight consecutive passages of 43 SARS-CoV-2 isolates in the presence of ciclesonide generated 15 resistant mutants harboring single amino acid substitutions in nonstructural protein 3 (nsp3) or nsp4. Of note, ciclesonide suppressed the replication of all these mutants by 90% or more, suggesting that these mutants cannot completely overcome ciclesonide blockade. Under a microscope, the viral RNA replication-transcription complex in cells, which is thought to be detectable using antibodies specific for nsp3 and double-stranded RNA, was observed to fall in the presence of ciclesonide in a concentration-dependent manner. These observations indicate that the suppressive effect of ciclesonide on viral replication is specific to coronaviruses, highlighting it as a candidate drug for the treatment of COVID-19 patients. IMPORTANCE The outbreak of SARS-CoV-2, the cause of COVID-19, is ongoing. New and effective antiviral agents that combat the disease are needed urgently. Here, we found that an inhaled corticosteroid, ciclesonide, suppresses the replication of coronaviruses, including betacoronaviruses (murine hepatitis virus type 2 [MHV-2], MERS-CoV, SARS-CoV, and SARS-CoV-2) and an alphacoronavirus (human coronavirus 229E [HCoV-229E]), in cultured cells. Ciclesonide is safe; indeed, it can be administered to infants at high concentrations. Thus, ciclesonide is expected to be a broad-spectrum antiviral drug that is effective against many members of the coronavirus family. It could be prescribed for the treatment of MERS and COVID-19.

scholarly journals A Dimerization-Dependent Mechanism Drives the Endoribonuclease Function of Porcine Reproductive and Respiratory Syndrome Virus nsp11

2016 ◽  
Vol 90 (9) ◽  
pp. 4579-4592 ◽  
Author(s):  
Yuejun Shi ◽  
Youwen Li ◽  
Yingying Lei ◽  
Gang Ye ◽  
Zhou Shen ◽  
...  
Keyword(s):  
Active Site ◽  
Antiviral Drug ◽  
Vital Role ◽  
Structural Basis ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Active Site Residues ◽  
Swine Industry ◽  
Content Type ◽  
Dimeric Structure

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) RNA endoribonuclease nsp11 belongs to the XendoU superfamily and plays a crucial role in arterivirus replication. Here, we report the first crystal structure of the arterivirus nsp11 protein from PRRSV, which exhibits a unique structure and assembles into an asymmetric dimer whose structure is completely different from the hexameric structure of coronavirus nsp15. However, the structures of the PRRSV nsp11 and coronavirus nsp15 catalytic domains were perfectly superimposed, especially in the “active site loop” (His129 to His144) and “supporting loop” (Val162 to Thr179) regions. Importantly, our biochemical data demonstrated that PRRSV nsp11 exists mainly as a dimer in solution. Mutations of the major dimerization site determinants (Ser74 and Phe76) in the dimerization interface destabilized the dimer in solution and severely diminished endoribonuclease activity, indicating that the dimer is the biologically functional unit. In the dimeric structure, the active site loop and supporting loop are packed against one another and stabilized by monomer-monomer interactions. These findings may help elucidate the mechanism underlying arterivirus replication and may represent great potential for the development of antiviral drugs.IMPORTANCEPorcine reproductive and respiratory syndrome virus (PRRSV) is a member of the familyArteriviridae, orderNidovirales. PRRSV is a major agent of respiratory diseases in pigs, causing tremendous economic losses to the swine industry worldwide. The PRRSV nsp11 endoribonuclease plays a vital role in arterivirus replication, but its precise roles and mechanisms of action are poorly understood. Here, we report the first dimeric structure of the arterivirus nsp11 from PRRSV at 2.75-Å resolution. Structural and biochemical experiments demonstrated that nsp11 exists mainly as a dimer in solution and that nsp11 may be fully active as a dimer. Mutagenesis and structural analysis revealed NendoU active site residues, which are conserved throughout the orderNidovirales(familiesArteriviridaeandCoronaviridae) and the major determinants of dimerization (Ser74 and Phe76) inArteriviridae. Importantly, these findings may provide a new structural basis for antiviral drug development.

scholarly journals Intracellular Free Iron and Its Potential Role in Ultrahigh-Pressure-Induced Inactivation of Escherichia coli

2012 ◽  
Vol 79 (2) ◽  
pp. 722-724 ◽  
Author(s):  
Yuan Yan ◽  
Joy G. Waite-Cusic ◽  
Periannan Kuppusamy ◽  
Ahmed E. Yousef
Keyword(s):  
Escherichia Coli ◽  
Iron Chelator ◽  
Ultrahigh Pressure ◽  
Dependent Manner ◽  
Paramagnetic Resonance ◽  
Free Iron ◽  
Content Type ◽  
E Coli ◽  
Electron Paramagnetic ◽  
Dose Dependent

ABSTRACTIntracellular free iron ofEscherichia coliwas determined by whole-cell electron paramagnetic resonance spectrometry. Ultrahigh pressure (UHP) increased both intracellular free iron and cell lethality in a pressure-dose-dependent manner. The iron chelator 2,2′-dipyridyl protected cells against UHP treatments. A mutation that produced iron overload conditions sensitizedE. colito UHP treatment.

scholarly journals Porcine Reproductive and Respiratory Syndrome Virus Induces IL-1βProduction Depending on TLR4/MyD88 Pathway and NLRP3 Inflammasome in Primary Porcine Alveolar Macrophages

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Jing Bi ◽  
Shuang Song ◽  
Liurong Fang ◽  
Dang Wang ◽  
Huiyuan Jing ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Alveolar Macrophages ◽  
Nlrp3 Inflammasome ◽  
Respiratory Syndrome Virus ◽  
Dependent Manner ◽  
Swine Industry ◽  
Downstream Signaling ◽  
Infection Treatment ◽  
Sirna Knockdown ◽  
Myd88 Pathway

Porcine reproductive and respiratory syndrome virus (PRRSV) is anArterivirusthat has been devastating the swine industry worldwide since the late 1980s. Previous studies have reported that PRRSV infection induced the production of IL-1β. However, the cellular sensors and signaling pathways involved in this process have not been elucidated yet. Here, we studied the mechanisms responsible for the production of IL-1βin response to highly pathogenic PRRSV. Upon PRRSV infection of primary porcine alveolar macrophages, both mRNA expression and secretion of IL-1βwere significantly increased in a time- and dose-dependent manner. We also investigated the role of several pattern-recognition receptors and adaptor molecules in this response and showed that the TLR4/MyD88 pathway and its downstream signaling molecules, NF-κB, ERK1/2, and p38 MAPKs, were involved in IL-1βproduction during PRRSV infection. Treatment with specific inhibitors or siRNA knockdown assays demonstrated that components of the NLRP3 inflammasome were crucial for IL-1βsecretion but not for IL-1βmRNA expression. Furthermore, TLR4/MyD88/NF-κB signaling pathway was involved in PRRSV-induced expression of NLRP3 inflammasome components. Together, our results deciphered the pathways leading from recognition of PRRSV to the production and release of IL-1β, providing a deeper knowledge of the mechanisms of PRRSV-induced inflammation responses.

scholarly journals Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity

2001 ◽  
Vol 75 (22) ◽  
pp. 10651-10662 ◽  
Author(s):  
Lin Qu ◽  
Laura K. McMullan ◽  
Charles M. Rice
Keyword(s):  
Cultured Cells ◽  
Nonstructural Protein ◽  
Low Frequency ◽  
Endoplasmic Reticulum Membrane ◽  
Diarrhea Virus ◽  
Isolation And Characterization ◽  
Cytosolic Side ◽  
Infected Cells ◽  
Dominant Selectable Marker ◽  
Viral Diarrhea Virus

ABSTRACT Isolates of bovine viral diarrhea virus (BVDV), the prototype pestivirus, are divided into cytopathic (cp) and noncytopathic (ncp) biotypes according to their effect on cultured cells. The cp viruses also differ from ncp viruses by the production of viral nonstructural protein NS3. However, the mechanism by which cp viruses induce cytopathic effect in cell culture remains unknown. Here we used a genetic approach to isolate ncp variants that arose from a cp virus at low frequency. A bicistronic BVDV (cp strain NADL) was created that expressed puromycin acetyltransferase as a dominant selectable marker. This bicistronic virus exhibited slightly slower growth kinetics and smaller plaques than NADL but remained cp. A number of independent ncp variants were isolated by puromycin selection. Remarkably, these ncp variants produced NS3 and viral RNA at levels comparable to those of the cp parent. Sequence analyses uncovered no change in NS3, but for all ncp variants a Y2441C substitution at residue 15 of NS4B was found. Introduction of the Y2441C substitution into the NADL or bicistronic cp viruses reconstituted the ncp phenotype. Y2441 is highly conserved among pestiviruses and is located in a region of NS4B predicted to be on the cytosolic side of the endoplasmic reticulum membrane. Other engineered substitutions for Y2441 also affected viral cytopathogenicity and viability, with Y2441V being cp, Y2441A being ncp, and Y2441D rendering the virus unable to replicate. The ncp substitutions for Y2441 resulted in slightly increased levels of NS2-3 relative to NS3. We also showed that NS3, NS4B, and NS5A could be chemically cross-linked in NADL-infected cells, indicating that they are associated as components of a multiprotein complex. Although the mechanism remains to be elucidated, these results demonstrate that mutations in NS4B can attenuate BVDV cytopathogenicity despite NS3 production.

scholarly journals Dynamics of Coronavirus Replication-Transcription Complexes

2009 ◽  
Vol 84 (4) ◽  
pp. 2134-2149 ◽  
Author(s):  
Marne C. Hagemeijer ◽  
Monique H. Verheije ◽  
Mustafa Ulasli ◽  
Indra A. Shaltiël ◽  
Lisa A. de Vries ◽  
...  
Keyword(s):  
Live Cell Imaging ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Dependent Manner ◽  
C Terminus ◽  
Efficient Replication ◽  
Infected Cells ◽  
Biochemical Analyses ◽  
Transcription Complexes ◽  
Cytoplasmic Side

ABSTRACT Coronaviruses induce in infected cells the formation of double-membrane vesicles (DMVs) in which the replication-transcription complexes (RTCs) are anchored. To study the dynamics of these coronavirus replicative structures, we generated recombinant murine hepatitis coronaviruses that express tagged versions of the nonstructural protein nsp2. We demonstrated by using immunofluorescence assays and electron microscopy that this protein is recruited to the DMV-anchored RTCs, for which its C terminus is essential. Live-cell imaging of infected cells demonstrated that small nsp2-positive structures move through the cytoplasm in a microtubule-dependent manner. In contrast, large fluorescent structures are rather immobile. Microtubule-mediated transport of DMVs, however, is not required for efficient replication. Biochemical analyses indicated that the nsp2 protein is associated with the cytoplasmic side of the DMVs. Yet, no recovery of fluorescence was observed when (part of) the nsp2-positive foci were bleached. This result was confirmed by the observation that preexisting RTCs did not exchange fluorescence after fusion of cells expressing either a green or a red fluorescent nsp2. Apparently, nsp2, once recruited to the RTCs, is not exchanged with nsp2 present in the cytoplasm or at other DMVs. Our data show a remarkable resemblance to results obtained recently by others with hepatitis C virus. The observations point to intriguing and as yet unrecognized similarities between the RTC dynamics of different plus-strand RNA viruses.

scholarly journals Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen

2016 ◽  
Vol 23 (6) ◽  
pp. 460-469 ◽  
Author(s):  
Rúbens Prince dos Santos Alves ◽  
Lennon Ramos Pereira ◽  
Denicar Lina Nascimento Fabris ◽  
Felipe Scassi Salvador ◽  
Robert Andreata Santos ◽  
...  
Keyword(s):  
Immune Responses ◽  
Dengue Virus ◽  
Nonstructural Protein ◽  
Vaccine Antigen ◽  
Dependent Manner ◽  
Nonstructural Proteins ◽  
Necrosis Factor Alpha ◽  
Content Type ◽  
Factor Alpha ◽  
Dengue Virus Serotypes

ABSTRACTDengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed inEscherichia coliat large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses.

scholarly journals Hcp Family Proteins Secreted via the Type VI Secretion System Coordinately Regulate Escherichia coli K1 Interaction with Human Brain Microvascular Endothelial Cells

2011 ◽  
Vol 80 (3) ◽  
pp. 1243-1251 ◽  
Author(s):  
Yan Zhou ◽  
Jing Tao ◽  
Hao Yu ◽  
Jinjing Ni ◽  
Lingbing Zeng ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Human Brain ◽  
Microvascular Endothelial Cells ◽  
Dependent Manner ◽  
Brain Microvascular Endothelial Cells ◽  
Content Type ◽  
E Coli ◽  
Type Vi Secretion ◽  
Microvascular Endothelial ◽  
Cytoskeleton Rearrangement

Type VI secretion systems (T6SSs) are involved in the pathogenicity of several Gram-negative bacteria. Based on sequence analysis, we found that a cluster ofEscherichia colivirulencefactors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causingE. coliK1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ inE. coli-HBMEC interaction. The deletion mutant ofhcp2showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional inE. coliK1, and two Hcp family proteins participate in different steps ofE. coliinteraction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causingE. coliK1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis ofE. colimeningitis.

scholarly journals Gut Symbionts from Distinct Hosts Exhibit Genotoxic Activity via Divergent Colibactin Biosynthesis Pathways

2014 ◽  
Vol 81 (4) ◽  
pp. 1502-1512 ◽  
Author(s):  
Philipp Engel ◽  
Maria I. Vizcaino ◽  
Jason M. Crawford
Keyword(s):  
Dna Damage ◽  
Secondary Metabolites ◽  
Biosynthetic Pathway ◽  
Tumor Formation ◽  
Microbial Interactions ◽  
Eukaryotic Cells ◽  
Dependent Manner ◽  
Bacterial Symbionts ◽  
Content Type ◽  
E Coli

ABSTRACTSecondary metabolites produced by nonribosomal peptide synthetase (NRPS) or polyketide synthase (PKS) pathways are chemical mediators of microbial interactions in diverse environments. However, little is known about their distribution, evolution, and functional roles in bacterial symbionts associated with animals. A prominent example is colibactin, a largely unknown family of secondary metabolites produced byEscherichia colivia a hybrid NRPS-PKS biosynthetic pathway that inflicts DNA damage upon eukaryotic cells and contributes to colorectal cancer and tumor formation in the mammalian gut. Thus far, homologs of this pathway have only been found in closely relatedEnterobacteriaceae, while a divergent variant of this gene cluster was recently discovered in a marine alphaproteobacterialPseudovibriostrain. Herein, we sequenced the genome ofFrischella perraraPEB0191, a bacterial gut symbiont of honey bees and identified a homologous colibactin biosynthetic pathway related to those found inEnterobacteriaceae. We show that the colibactin genomic island (GI) has conserved gene synteny and biosynthetic module architecture acrossF. perrara,Enterobacteriaceae, and thePseudovibriostrain. Comparative metabolomics analyses ofF. perraraandE. colifurther reveal that these two bacteria produce related colibactin pathway-dependent metabolites. Finally, we demonstrate thatF. perrara, likeE. coli, causes DNA damage in eukaryotic cellsin vitroin a colibactin pathway-dependent manner. Together, these results support that divergent variants of the colibactin biosynthetic pathway are widely distributed among bacterial symbionts, producing related secondary metabolites and likely endowing its producer with functional capabilities important for diverse symbiotic associations.

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