Desialylation by Edwardsiella tarda is the initial step in the regulation of its invasiveness

2019 ◽  
Vol 476 (21) ◽  
pp. 3183-3196
Author(s):  
Linh Khanh Vo ◽  
Toshiharu Tsuzuki ◽  
Yuko Kamada-Futagami ◽  
Petros Kingstone Chigwechokha ◽  
Akinobu Honda ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Mrna Level ◽  
Initial Step ◽  
Host Cells ◽  
Edwardsiella Tarda ◽  
Economic Losses ◽  
Mrna Levels ◽  
Sialidase Activity ◽  
Sialidase Inhibitor ◽  
The Relationship

Abstract Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from α2–3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

scholarly journals Altered interaction between RBD and ACE2 receptor contributes towards the increased transmissibility of SARS CoV-2 delta, kappa, beta, and gamma strains with RBD double mutations

2021 ◽  
Author(s):  
Siddharth Sinha ◽  
Benjamin Tam ◽  
San Ming Wang
Keyword(s):  
Initial Step ◽  
Host Cells ◽  
Dynamics Simulation ◽  
Single Mutation ◽  
Host Cell Membrane ◽  
Energy Estimation ◽  
Double Mutation ◽  
Free Binding Energy ◽  
The Relationship ◽  
New Mutations

ABSTRACTThe COVID-19 pandemics by SARS-CoV-2 causes catastrophic damage for global human health. The initial step of SARS-CoV-2 infection is the binding of the receptor-binding domain (RBD) in its spike protein to ACE2 receptor in host cell membrane. The evolving of SARS-CoV-2 constantly generates new mutations across its genome including RBD. Besides the well-known single mutation in RBD, the recent new mutation strains with RBD “double mutation” is causing new outbreaks globally, as represented by the delta strain containing RBD L452R/T478K. Although it is considered that the increased transmissibility of the double mutated strains could be attributed to the alteration of mutated RBD to ACE2 receptor, the molecular details remains to be unclear. Using the methods of molecular dynamics simulation, superimposed structural comparison, free binding energy estimation and antibody escaping, we investigated the relationship between ACE2 receptor and the RBD double mutant L452R/T478K (delta), L452R/E484Q (kappa) and E484K/N501Y (beta, gamma). The results demonstrated that each of the three RBD double mutants altered RBD structure, led to enhanced binding affinity of mutated RBD to ACE2 receptor, leading to increased transmissibility of SARS-CoV-2 to the host cells.

Inhibition of Sialic Acid Loss Greatly Improves Survival of Refrigerated Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1133-1133
Author(s):  
Gerard Jansen ◽  
Emma C Josefsson ◽  
Qiyong Peter Liu ◽  
Viktoria Rumjantseva ◽  
Herve Falet ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Sodium Salt ◽  
Platelet Transfusion ◽  
Blood Components ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Sialidase Inhibitor ◽  
Rapid Clearance ◽  
Receptor Shedding ◽  
Von Willebrand

Abstract Abstract 1133 Platelets have the shortest shelf life of all major blood components and are the most difficult to store, a fact that complicates platelet transfusion practices. Platelet refrigeration could slow bacterial growth and possibly retard the loss of platelet function following storage. However, in contrast to other blood components, platelets do not tolerate refrigeration and are rapidly cleared from the circulation. We demonstrated that two distinct pathways recognizing GPIba remove refrigerated platelets in recipient's livers: 1) αMβ2 integrins (Mac-1) on hepatic resident macrophages (Kupffer cells) selectively recognize irreversibly clustered b-N-acetylglucosamine (β-GlcNAc)–terminated glycans on GPIbα, and 2) hepatic Asialoglycoprotein (Asg) receptors (Ashwell Morell receptors) recognize desialylated GPIba. We here investigated the mechanism of sialic acid loss during refrigeration. We show, that when refrigerated platelets are rewarmed, they secrete active sialidases, including the lysosomal sialidase Neu1 that remove sialic acid from platelet receptors, specifically from GPIbα. Platelets also express Neu3 on their surfaces, however Neu3 expression appears to be unaffected by platelet refrigeration. Importantly, the recovery and circulation of refrigerated platelets is greatly improved by storage in the presence of the competitive sialidase inhibitor N-Acetylneuraminic Acid, 2,3-Dehydro-2-deoxy-Sodium Salt (DANA). Desialylated von Willebrand receptor (vWfR) complex is also a target for metalloproteinases (MMPs), as GPIbα and GPV are cleaved from the surface of refrigerated platelets. Receptor shedding is inhibited by the metalloproteinase inhibitor GM6001 and does not occur in ADAM17ΔZn/ΔZn platelets expressing inactive ADAM17. Critically, desialylation in the absence of metalloproteinase-mediated receptor shedding is sufficient to induce the rapid clearance of platelets from circulation. Desialylation of platelet vWfR therefore triggers platelet clearance, and primes GPIbα and GPV for metalloproteinase-dependent cleavage. We conclude that desialylation of platelets is caused by increased surface sialidase activity following refrigeration and desialylation of glycoproteins, specifically of GPIbα, promotes receptor cleavage by MMPs. Disclosures: Liu: Velicomedical, Inc: Employment.

Effects of obesity on the relationship of leptin mRNA expression and adipocyte size in anatomically distinct fat depots in mice

2004 ◽  
Vol 287 (1) ◽  
pp. R112-R119 ◽  
Author(s):  
Kai-Ying Guo ◽  
Patricia Halo ◽  
Rudolph L. Leibel ◽  
Yiying Zhang
Keyword(s):  
Fat Mass ◽  
Mrna Level ◽  
Physiological Role ◽  
Mrna Levels ◽  
Obese Mice ◽  
Positive Correlation ◽  
Fat Depots ◽  
Adipocyte Volume ◽  
The Relationship ◽  
Young Mice

In support of leptin's physiological role as humoral signal of fat mass, we have shown that adipocyte volume is a predominant determinant of leptin mRNA levels in anatomically distinct fat depots in lean young mice in the postabsorptive state. In this report, we investigated how obesity may affect the relationship between leptin mRNA levels and adipocyte volume in anatomically distinct fat depots in mice with genetic ( Lep ob/ Lep ob and A y /+), diet-induced, and aging-related obesity. In all of the obese mice examined, tissue leptin mRNA levels relative to the average adipocyte volume were lower in the perigonadal and/or retroperitoneal than in the inguinal fat depots and were lower than those of the lean young mice in the perigonadal fat depot. A close, positive correlation between leptin mRNA level and adipocyte volume was present from small to hypertrophic adipocytes within each perigonadal and inguinal fat pad in the obese mice, but the slopes of the regression lines relating leptin mRNA level to adipocyte volume were significantly lower in the perigonadal than in the inguinal fat pads of the same mice. These results suggest that obesity per se is associated with a decreased leptin gene expression per unit of fat mass in mice and that the positive correlation between leptin mRNA level and adipocyte volume is an intrinsic property of adipocytes that is not disrupted by adipocyte hypertrophy in obese mice.

scholarly journals Role of sialidase in glycoprotein utilization by Tannerella forsythia

Microbiology ◽  
2011 ◽  
Vol 157 (11) ◽  
pp. 3195-3202 ◽  
Author(s):  
Sumita Roy ◽  
Kiyonobu Honma ◽  
C. W. Ian Douglas ◽  
Ashu Sharma ◽  
Graham P. Stafford
Keyword(s):  
Sialic Acid ◽  
Tannerella Forsythia ◽  
Similar Reduction ◽  
Biofilm Growth ◽  
Sialidase Activity ◽  
Sialidase Inhibitor ◽  
Initial Adhesion ◽  
Coated Surfaces

The major bacterial pathogens associated with periodontitis include Tannerella forsythia. We previously discovered that sialic acid stimulates biofilm growth of T. forsythia, and that sialidase activity is key to utilization of sialoconjugate sugars and is involved in host–pathogen interactions in vitro. The aim of this work was to assess the influence of the NanH sialidase on initial biofilm adhesion and growth in experiments where the only source of sialic acid was sialoglycoproteins or human oral secretions. After showing that T. forsythia can utilize sialoglycoproteins for biofilm growth, we showed that growth and initial adhesion with sialylated mucin and fetuin were inhibited two- to threefold by the sialidase inhibitor oseltamivir. A similar reduction (three- to fourfold) was observed with a nanH mutant compared with the wild-type. Importantly, these data were replicated using clinically relevant serum and saliva samples as substrates. In addition, the ability of the nanH mutant to form biofilms on glycoprotein-coated surfaces could be restored by the addition of purified NanH, which we show is able to cleave sialic acid from the model glycoprotein fetuin and, much less efficiently, 9-O-acetylated bovine submaxillary mucin. These data show for the first time that glycoprotein-associated sialic acid is likely to be a key in vivo nutrient source for T. forsythia when growing in a biofilm, and suggest that sialidase inhibitors might be useful adjuncts in periodontal therapy.

Sialic Acid Loss Regulates Platelet Survival and Integrity

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3504-3504
Author(s):  
Renata Grozovsky ◽  
Gerard Jansen ◽  
Karin M. Hoffmeister
Keyword(s):  
Flow Cytometry ◽  
Sialic Acid ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Platelet Survival ◽  
Sialidase Inhibitor ◽  
Glycan Degradation

Abstract It becomes increasingly apparent that, besides the intrinsic apoptotic machinery, surface glycan modifications regulate platelet survival. Platelets with reduced α2,3-linked sialic acid during sepsis due to S. pneumoniae infection, after cold storage, or in mice lacking the sialyltransferase ST3GalIV are cleared by the hepatic Ashwell-Morell receptor (AMR, ASGPR1/2). Platelet survival in Asgr2-/- mice was increased by ∼35% when compared to that of WT mice, which results in a ∼50% increase in circulating platelet counts, despite a loss of surface sialic acid. We reasoned that sialidase activity increases on the surface of circulating platelets as they age, a process that would facilitate sialic acid hydrolysis and removal from the circulation. To test this hypothesis, we directly injected the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) into WT mice and determined endogenous platelet circulatory times. Platelet survival was prolonged by ∼30% (T1/2 of 62.0 ± 2.7 h) in DANA-treated mice, compared to that of mock-treated mice (T1/2 of 47.5 ± 4.3 h). DANA injections decreased terminal sialic acid loss on circulating platelets by ∼40% by day 2, compared to control platelets, as evidenced by binding of RCA-I lectin that specifically recognizes terminal β1-4 galactose moieties exposed by sialic acid removal. Freshly isolated, resting platelets from Asgr2-/- mice (AMR-platelets) were significantly smaller in size (22%) and had increased sialidase Neu1 (∼5 fold), but not Neu3 surface expression, when compared to WT platelets or St3gal4-/- platelets, as measured by flow cytometry. We next investigated if AMR-platelets age/deteriorate faster upon in vitro storage. Platelets were isolated from WT, Asgr2-/- and St3gal4-/- mice and stored for 24hrs at room temperature, and sialidase expression (Neu1 and Neu3) as well as microvesiculation were measured by flow cytometry. Although significant Neu1 and Neu3 surface expression increase was measured on platelets from all phenotype after storage, Neu1 and Neu3 surface expression was significantly higher in AMR-platelets (∼2 and 4 fold, respectively) when compared to WT and St3gal4-/- platelets. AMR-platelets, but not St3gal4-/- platelets microvesiculated upon storage, consistent with a faster deterioration of aged AMR-platelets. We next injected into WT and Asgr2-/- mice the BH3 mimetic, ABT-737, which binds and inhibits the pro-apoptotic Bcl-2, Bcl-xL and Bcl-w. After injection of ABT-737, platelets in the Asgr2-/- mouse were cleared more efficiently (∼20%) from the circulation when compared to those in WT mice. Collectively, our data show that blood borne sialidases contribute to loss of sialic acid during circulation to regulate platelet survival. Our data also suggest that platelet glycan degradation, i.e. sialic acid loss, may trigger the intrinsic apoptotic machinery in platelets, linking glycan degradation and intrinsic apoptotic machinery in the clearance mechanisms regulating platelet survival. Disclosures: No relevant conflicts of interest to declare.

MiR-493 Induces Cytotoxic Autophagy in Prostate Cancer Cells through Regulation on PHLPP2

2020 ◽  
Vol 21 (14) ◽  
pp. 1451-1456 ◽  
Author(s):  
Jun Deng ◽  
Ming Ma ◽  
Wei Jiang ◽  
Liangliang Zheng ◽  
Suping Cui
Keyword(s):  
Prostate Cancer ◽  
Protein Expression ◽  
Cell Function ◽  
Mrna Levels ◽  
Prostate Cancer Cells ◽  
Potent Inducer ◽  
Qrt Pcr ◽  
Autophagy Gene ◽  
The Relationship ◽  
Cancer Inhibition

Background: MiR-493 promotes the proliferation of prostate cancer (PC) cells by targeting PHLPP2. We aimed to explore the relationship between miR-493 and autophagy in PC. Methods: qRT-PCR and western blotting were used to determine the mRNA levels and protein expression of miR-493, PHLPP2, autophagy gene BECN1 and ATG7 in PC cells. The autophagy gene expression was determined after PC cells transfected with miR-493 precursor or PHLPP2 precursor. Corresponding changes of autophagy phenotype and PC cell function were also studied. Results: The mRNA levels and protein expression of miR-493, PHLPP2, BECN1 and ATG7 in PC cells were significantly decreased in PC cells. Overexpression of miR-493 or PHLPP2 markedly upregulated the expression levels of BECN1 and ATG7 in PC cells. Overexpression of miR-493 and PHLPP2 markedly promoted autophagy, and inhibited the invasion and cloning formation of PC cells. Conclusion: MiR-493 is a potent inducer of cytotoxic autophagy that leads to prostate cancer inhibition by regulating on PHLPP2.

HIF1A is Overexpressed in Medulloblastoma and its Inhibition Reduces Proliferation and Increases EPAS1 and ATG16L1 Methylation

2018 ◽  
Vol 18 (3) ◽  
pp. 287-294 ◽  
Author(s):  
Gustavo Alencastro Veiga Cruzeiro ◽  
Maristella Bergamo dos Reis ◽  
Vanessa Silva Silveira ◽  
Regia Caroline Peixoto Lira ◽  
Carlos Gilberto Carlotti Jr ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Cell Line ◽  
Cellular Proliferation ◽  
Mrna Level ◽  
Relevant Information ◽  
Protein Stabilization ◽  
Mrna Levels ◽  
Medulloblastoma Cell Line ◽  
Pediatric Medulloblastoma ◽  
Fresh Frozen

Background: Genetic and epigenetic modifications are closely related to tumor initiation and progression and can provide guidance for understanding tumor functioning, potentially leading to the discovery of new therapies. Studies have associated hypoxia-related genes to tumor progression and chemo/radioresistance in brain tumors. Information on the expression profile of hypoxiarelated genes in pediatric medulloblastoma, although scarce, may reveal relevant information that could support treatment decisions. Objective: Our study focused on evaluation the of CA9, CA12, HIF1A, EPAS1, SCL2A1 and VEGF genes in 41 pediatric fresh-frozen medulloblastoma sample. Additionally, we analyzed the effect of hypoxia and normoxia in the pediatric medulloblastoma cell-line UW402. Furthermore, we assessed the effects of HIF1A knockdown in cell-proliferation and methylation levels of genes related to hypoxia, apoptosis and autophagy. Method: qPCR was performed to evaluate mRNA levels, and Western blot to confirm HIF1A silencing in both patient samples and cell line. Pyrosequencing was performed to asses the methylation levels after HIF1A knockdown in the UW402 cell line. Results: A higher HIF1A mRNA level was observed in MB patients when compared to the cerebellum (non-tumor match). In UW402 MB cell-line, chemically induced hypoxic resulted in an increase of mRNA levels of HIF1A, VEGF, SCL2A1 and CA9 genes. Additionally, HIF1A knockdown induced a decrease in the expression of hypoxia related genes and a decrease of 30% in cell proliferation was also observed. Also, a significant increase in the methylation of ATG16L1 promoter and decrease in the methylation of EPAS1 promoter were observed after HIF1A knockdown. Conclusion: HIF1A knockdown in medulloblastoma cells lead to decreased cellular proliferation, suggesting that HIF1A can be a potential therapeutic target to be explored in the medulloblastoma. However, the mechanisms behind HIF1A protein stabilization and function are very complex and more data need to be generated to potentially use HIF1A as a therapeutical target.

scholarly journals Enhancement of elastin expression by transdermal administration of sialidase isozyme Neu2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Akira Minami ◽  
Yuka Fujita ◽  
Jun Goto ◽  
Ayano Iuchi ◽  
Kosei Fujita ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Skin Diseases ◽  
Lower Layer ◽  
Elastic Fiber ◽  
Sialic Acid Residue ◽  
Elastic Fibers ◽  
Transdermal Administration ◽  
Sialidase Activity ◽  
Fiber Assembly ◽  
Senescence Accelerated Mice

AbstractReduction of elastin in the skin causes various skin diseases as well as wrinkles and sagging with aging. Sialidase is a hydrolase that cleaves a sialic acid residue from sialoglycoconjugate. Cleavage of sialic acid from microfibrils by the sialidase isozyme Neu1 facilitates elastic fiber assembly. In the present study, we showed that a lower layer of the dermis and muscle showed relatively intense sialidase activity. The sialidase activity in the skin decreased with aging. Choline and geranate (CAGE), one of the ionic liquids, can deliver the sialidase subcutaneously while maintaining the enzymatic activity. The elastin level in the dermis was increased by applying sialidase from Arthrobacter ureafaciens (AUSA) with CAGE on the skin for 5 days in rats and senescence-accelerated mice prone 1 and 8. Sialidase activity in the dermis was considered to be mainly due to Neu2 based on the expression level of sialidase isozyme mRNA. Transdermal administration of Neu2 with CAGE also increased the level of elastin in the dermis. Therefore, not only Neu1 but also Neu2 would be involved in elastic fiber assembly. Transdermal administration of sialidase is expected to be useful for improvement of wrinkles and skin disorders due to the loss of elastic fibers.

scholarly journals Cell Culture-Derived Tilapia Lake Virus-Inactivated Vaccine Containing Montanide Adjuvant Provides High Protection against Viral Challenge for Tilapia

Vaccines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 86
Author(s):  
Weiwei Zeng ◽  
Yingying Wang ◽  
Huzi Hu ◽  
Qing Wang ◽  
Sven M. Bergmann ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Protective Efficacy ◽  
Interferon Γ ◽  
Economic Losses ◽  
Mrna Levels ◽  
Infectious Dose ◽  
Booster Immunization ◽  
Virus Challenge ◽  
High Level ◽  
Factor Α

Tilapia lake virus (TiLV) is a newly emerging pathogen responsible for high mortality and economic losses in the global tilapia industry. Currently, no antiviral therapy or vaccines are available for the control of this disease. The goal of the present study was to evaluate the immunological effects and protective efficacy of formaldehyde- and β-propiolactone-inactivated vaccines against TiLV in the presence and absence of the Montanide IMS 1312 VG adjuvant in tilapia. We found that β-propiolactone inactivation of viral particles generated a vaccine with a higher protection efficacy against virus challenge than did formaldehyde. The relative percent survivals of vaccinated fish at doses of 108, 107, and 106 50% tissue culture infectious dose (TCID50)/mL were 42.9%, 28.5%, and 14.3% in the absence of the adjuvant and 85.7%, 64.3%, and 32.1% in its presence, respectively. The vaccine generated specific IgM and neutralizing antibodies against TiLV at 3 weeks following immunization that were significantly increased after a second booster immunization. The steady state mRNA levels of the genes tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon γ (IFN-γ), cluster of differentiation 4 (CD4), major histocompatibility complex (MHC)-Ia, and MHC-II were all increased and indicated successful immune stimulation against TiLV. The vaccine also significantly lowered the viral loads and resulted in significant increases in survival, indicating that the vaccine may also inhibit viral proliferation as well as stimulate a protective antibody response. The β-propiolactone-inactivated TiLV vaccine coupled with the adjuvant Montanide IMS 1312 VG and booster immunizations can provide a high level of protection from virus challenge in tilapia.

scholarly journals Role of ASM/Cer/TXNIP signaling module in the NLRP3 inflammasome activation

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Jianjun Jiang ◽  
Yining Shi ◽  
Jiyu Cao ◽  
Youjin Lu ◽  
Gengyun Sun ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Nlrp3 Inflammasome ◽  
Mrna Level ◽  
Scavenger Receptor ◽  
Nuclear Factor Κb ◽  
Inflammasome Activation ◽  
Mrna Levels ◽  
Irreversible Inhibitor ◽  
Nlrp3 Inflammasome Activation

Abstract Background This study aimed to explore the effects of ceramide (Cer) on NLRP3 inflammasome activation and their underlying mechanisms. Methods Lipopolysaccharide (LPS)/adenosine triphosphate (ATP)-induced NLRP3 inflammasome activation in J774A.1 cells and THP-1 macrophages was used as an in vitro model of inflammation. Western blotting and real-time PCR (RT-PCR) were used to detect the protein and mRNA levels, respectively. IL-1β and IL-18 levels were measured by ELISA. ASM assay kit and immunofluorescence were used to detect ASM activity and Cer content. Results Imipramine, a well-known inhibitor of ASM, significantly inhibited LPS/ATP-induced activity of ASM and the consequent accumulation of Cer. Additionally, imipramine suppressed the LPS/ATP-induced expression of thioredoxin interacting protein (TXNIP), NLRP3, caspase-1, IL-1β, and IL-18 at the protein and mRNA level. Interestingly verapamil, a TXNIP inhibitor, suppressed LPS/ATP-induced activation of TXNIP/NLRP3 inflammasome but did not affect LPS/ATP-induced ASM activation and Cer formation. TXNIP siRNA and verapamil inhibited C2-Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome. In addition, the pretreatment of cells with sulfo-N-succinimidyl oleate (SSO), an irreversible inhibitor of the scavenger receptor CD36, blocked Cer-induced upregulation of nuclear factor-κB (NF-κB) activity, TXNIP expression, and NLRP3 inflammasome activation. Inhibition of NF-κB activation by SN50 prevented Cer-induced upregulation of TXNIP and activation of the NLRP3 inflammasome but did not affect CD36 expression. Conclusion This study demonstrated that the ASM/Cer/TXNIP signaling pathway is involved in NLRP3 inflammasome activation. The results documented that the CD36-dependent NF-κB-TXNIP signaling pathway plays an essential role in the Cer-induced activation of NLRP3 inflammasomes in macrophages.

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