136 results found
Griffith S, Muir L, Suchanek O, et al., 2024, Preservation of memory B cell homeostasis in an individual producing broadly neutralising antibodies against HIV-1., bioRxiv
Immunological determinants favouring emergence of broadly neutralising antibodies are crucial to the development of HIV-1 vaccination strategies. Here, we combined RNAseq and B cell cloning approaches to isolate a broadly neutralising antibody (bnAb) ELC07 from an individual living with untreated HIV-1. Using single particle cryogenic electron microscopy (cryo-EM), we show that the antibody recognises a conformational epitope at the gp120-gp41 interface. ELC07 binds the closed state of the viral glycoprotein causing considerable perturbations to the gp41 trimer core structure. Phenotypic analysis of memory B cell subsets from the ELC07 bnAb donor revealed a lack of expected HIV-1-associated dysfunction, specifically no increase in CD21 - /CD27 - cells was observed whilst the resting memory (CD21 + /CD27 + ) population appeared preserved despite uncontrolled HIV-1 viraemia. Moreover, single cell transcriptomes of memory B cells from this bnAb donor showed a resting memory phenotype irrespective of the epitope they targeted or their ability to neutralise diverse strains of HIV-1. Strikingly, single memory B cells from the ELC07 bnAb donor were transcriptionally similar to memory B cells from HIV-negative individuals. Our results demonstrate that potent bnAbs can arise without the HIV-1-induced dysregulation of the memory B cell compartment and suggest that sufficient levels of antigenic stimulation with a strategically designed immunogen could be effective in HIV-negative vaccine recipients.
Dinh T, Tber Z, Rey JS, et al., 2024, The structural and mechanistic bases for the viral resistance to allosteric HIV-1 integrase inhibitor pirmitegravir., bioRxiv
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are investigational antiretroviral agents which potently impair virion maturation by inducing hyper-multimerization of IN and inhibiting its interaction with viral genomic RNA. The pyrrolopyridine-based ALLINI pirmitegravir (PIR) has recently advanced into Phase 2a clinical trials. Previous cell culture based viral breakthrough assays identified the HIV-1(Y99H/A128T IN) variant that confers substantial resistance to this inhibitor. Here, we have elucidated the unexpected mechanism of viral resistance to PIR. While both Tyr99 and Ala128 are positioned within the inhibitor binding V-shaped cavity at the IN catalytic core domain (CCD) dimer interface, the Y99H/A128T IN mutations did not substantially affect direct binding of PIR to the CCD dimer or functional oligomerization of full-length IN. Instead, the drug-resistant mutations introduced a steric hindrance at the inhibitor mediated interface between CCD and C-terminal domain (CTD) and compromised CTD binding to the CCDY99H/A128T + PIR complex. Consequently, full-length INY99H/A128T was substantially less susceptible to the PIR induced hyper-multimerization than the WT protein, and HIV-1(Y99H/A128T IN) conferred >150-fold resistance to the inhibitor compared to the WT virus. By rationally modifying PIR we have developed its analog EKC110, which readily induced hyper-multimerization of INY99H/A128T in vitro and was ~14-fold more potent against HIV-1(Y99H/A128T IN) than the parent inhibitor. These findings suggest a path for developing improved PIR chemotypes with a higher barrier to resistance for their potential clinical use.
Ren R, Cai S, Fang X, et al., 2023, Multiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes, Nature Communications, Vol: 14, ISSN: 2041-1723
We report on single-molecule nanopore sensing combined with position-encoded DNA molecular probes, with chemistry tuned to simultaneously identify various antigen proteins and multiple RNA gene fragments of SARS-CoV-2 with high sensitivity and selectivity. We show that this sensing strategy can directly detect spike (S) and nucleocapsid (N) proteins in unprocessed human saliva. Moreover, our approach enables the identification of RNA fragments from patient samples using nasal/throat swabs, enabling the identification of critical mutations such as D614G, G446S, or Y144del among viral variants. In particular, it can detect and discriminate between SARS-CoV-2 lineages of wild-type B.1.1.7 (Alpha), B.1.617.2 (Delta), and B.1.1.539 (Omicron) within a single measurement without the need for nucleic acid sequencing. The sensing strategy of the molecular probes is easily adaptable to other viral targets and diseases and can be expanded depending on the application required.
Chiduza GN, Garza-Garcia A, Almacellas E, et al., 2023, ATG9B is a tissue-specific homotrimeric lipid scramblase that can compensate for ATG9A., Autophagy, Pages: 1-20
Macroautophagy/autophagy is a fundamental aspect of eukaryotic biology, and the autophagy-related protein ATG9A is part of the core machinery facilitating this process. In addition to ATG9A vertebrates encode ATG9B, a poorly characterized paralog expressed in a subset of tissues. Herein, we characterize the structure of human ATG9B revealing the conserved homotrimeric quaternary structure and explore the conformational dynamics of the protein. Consistent with the experimental structure and computational chemistry, we establish that ATG9B is a functional lipid scramblase. We show that ATG9B can compensate for the absence of ATG9A in starvation-induced autophagy displaying similar subcellular trafficking and steady-state localization. Finally, we demonstrate that ATG9B can form a heteromeric complex with ATG2A. By establishing the molecular structure and function of ATG9B, our results inform the exploration of niche roles for autophagy machinery in more complex eukaryotes and reveal insights relevant across species.Abbreviation: ATG: autophagy related; CHS: cholesteryl hemisuccinate; cryo-EM: single-particle cryogenic electron microscopy; CTF: contrast transfer function: CTH: C- terminal α helix; FSC: fourier shell correlation; HDIR: HORMA domain interacting region; LMNG: lauryl maltose neopentyl glycol; MD: molecular dynamics simulations; MSA: multiple sequence alignment; NBD-PE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl ammonium salt); POPC: palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; RBG: repeating beta groove domain; RMSD: root mean square deviation; SEC: size-exclusion chromatography; TMH: transmembrane helix.
Grandi A, Tomasi M, Ullah I, et al., 2023, Immunogenicity and Pre-Clinical Efficacy of an OMV-Based SARS-CoV-2 Vaccine., Vaccines (Basel), Vol: 11, ISSN: 2076-393X
The vaccination campaign against SARS-CoV-2 relies on the world-wide availability of effective vaccines, with a potential need of 20 billion vaccine doses to fully vaccinate the world population. To reach this goal, the manufacturing and logistic processes should be affordable to all countries, irrespective of economical and climatic conditions. Outer membrane vesicles (OMVs) are bacterial-derived vesicles that can be engineered to incorporate heterologous antigens. Given the inherent adjuvanticity, such modified OMVs can be used as vaccines to induce potent immune responses against the associated proteins. Here, we show that OMVs engineered to incorporate peptides derived from the receptor binding motif (RBM) of the spike protein from SARS-CoV-2 elicit an effective immune response in vaccinated mice, resulting in the production of neutralizing antibodies (nAbs) with a titre higher than 1:300. The immunity induced by the vaccine is sufficient to protect the animals from intranasal challenge with SARS-CoV-2, preventing both virus replication in the lungs and the pathology associated with virus infection. Furthermore, we show that OMVs can be effectively decorated with the RBM of the Omicron BA.1 variant and that such engineered OMVs induce nAbs against Omicron BA.1 and BA.5, as measured using the pseudovirus neutralization infectivity assay. Importantly, we show that the RBM438-509 ancestral-OMVs elicited antibodies which efficiently neutralize in vitro both the homologous ancestral strain, the Omicron BA.1 and BA.5 variants with a neutralization titre ranging from 1:100 to 1:1500, suggesting its potential use as a vaccine targeting diverse SARS-CoV-2 variants. Altogether, given the convenience associated with the ease of engineering, production and distribution, our results demonstrate that OMV-based SARS-CoV-2 vaccines can be a crucial addition to the vaccines currently available.
Baggen J, Jacquemyn M, Persoons L, et al., 2023, TMEM106B is a receptor mediating ACE2-independent SARS-CoV-2 cell entry, CELL, Vol: 186, Pages: 3427-+, ISSN: 0092-8674
Freeman SL, Oliveira ASF, Gallio AE, et al., 2023, Heme binding to the SARS-CoV-2 spike glycoprotein., J Biol Chem, Vol: 299
The target for humoral immunity, SARS-CoV-2 spike glycoprotein, has become the focus of vaccine research and development. Previous work demonstrated that the N-terminal domain (NTD) of SARS-CoV-2 spike binds biliverdin-a product of heme catabolism-causing a strong allosteric effect on the activity of a subset of neutralizing antibodies. Herein, we show that the spike glycoprotein is also able to bind heme (KD = 0.5 ± 0.2 μM). Molecular modeling indicated that the heme group fits well within the same pocket on the SARS-CoV-2 spike NTD. Lined by aromatic and hydrophobic residues (W104, V126, I129, F192, F194, I203, and L226), the pocket provides a suitable environment to stabilize the hydrophobic heme. Mutagenesis of N121 has a substantive effect on heme binding (KD = 3000 ± 220 μM), confirming the pocket as a major heme binding location of the viral glycoprotein. Coupled oxidation experiments in the presence of ascorbate indicated that the SARS-CoV-2 glycoprotein can catalyze the slow conversion of heme to biliverdin. The heme trapping and oxidation activities of the spike may allow the virus to reduce levels of free heme during infection to facilitate evasion of the adaptive and innate immunity.
Bonnard D, Le Rouzic E, Singer MR, et al., 2023, Biological and Structural Analyses of New Potent Allosteric Inhibitors of HIV-1 Integrase, ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Vol: 67, ISSN: 0066-4804
Powell AA, Ireland G, Leeson R, et al., 2023, National and regional prevalence of SARS-CoV-2 antibodies in primary and secondary school children in England: the School Infection Survey, a national open cohort study, November 2021SARS-CoV-2 antibody prevalence in school children, JOURNAL OF INFECTION, Vol: 86, Pages: 361-368, ISSN: 0163-4453
Parker E, Thomas J, Roper KJ, et al., 2023, SARS-CoV-2 antibody responses associate with sex, age and disease severity in previously uninfected people admitted to hospital with COVID-19: An ISARIC4C prospective study, Frontiers in Immunology, Vol: 14, Pages: 1-12, ISSN: 1664-3224
The SARS-CoV-2 pandemic enables the analysis of immune responses induced against a novel coronavirus infecting immunologically naïve individuals. This provides an opportunity for analysis of immune responses and associations with age, sex and disease severity. Here we measured an array of solid-phase binding antibody and viral neutralising Ab (nAb) responses in participants (n=337) of the ISARIC4C cohort and characterised their correlation with peak disease severity during acute infection and early convalescence. Overall, the responses in a Double Antigen Binding Assay (DABA) for antibody to the receptor binding domain (anti-RBD) correlated well with IgM as well as IgG responses against viral spike, S1 and nucleocapsid protein (NP) antigens. DABA reactivity also correlated with nAb. As we and others reported previously, there is greater risk of severe disease and death in older men, whilst the sex ratio was found to be equal within each severity grouping in younger people. In older males with severe disease (mean age 68 years), peak antibody levels were found to be delayed by one to two weeks compared with women, and nAb responses were delayed further. Additionally, we demonstrated that solid-phase binding antibody responses reached higher levels in males as measured via DABA and IgM binding against Spike, NP and S1 antigens. In contrast, this was not observed for nAb responses. When measuring SARS-CoV-2 RNA transcripts (as a surrogate for viral shedding) in nasal swabs at recruitment, we saw no significant differences by sex or disease severity status. However, we have shown higher antibody levels associated with low nasal viral RNA indicating a role of antibody responses in controlling viral replication and shedding in the upper airway. In this study, we have shown discernible differences in the humoral immune responses between males and females and these differences associate with age as well as with resultant disease severity.
Singer MR, Dinh T, Levintov L, et al., 2023, The Drug-Induced Interface That Drives HIV-1 Integrase Hypermultimerization and Loss of Function., mBio, Vol: 14
Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an emerging class of small molecules that disrupt viral maturation by inducing the aberrant multimerization of IN. Here, we present cocrystal structures of HIV-1 IN with two potent ALLINIs, namely, BI-D and the drug candidate Pirmitegravir. The structures reveal atomistic details of the ALLINI-induced interface between the HIV-1 IN catalytic core and carboxyl-terminal domains (CCD and CTD). Projecting from their principal binding pocket on the IN CCD dimer, the compounds act as molecular glue by engaging a triad of invariant HIV-1 IN CTD residues, namely, Tyr226, Trp235, and Lys266, to nucleate the CTD-CCD interaction. The drug-induced interface involves the CTD SH3-like fold and extends to the beginning of the IN carboxyl-terminal tail region. We show that mutations of HIV-1 IN CTD residues that participate in the interface with the CCD greatly reduce the IN-aggregation properties of Pirmitegravir. Our results explain the mechanism of the ALLINI-induced condensation of HIV-1 IN and provide a reliable template for the rational development of this series of antiretrovirals through the optimization of their key contacts with the viral target. IMPORTANCE Despite the remarkable success of combination antiretroviral therapy, HIV-1 remains among the major causes of human suffering and loss of life in poor and developing nations. To prevail in this drawn-out battle with the pandemic, it is essential to continue developing advanced antiviral agents to fight drug resistant HIV-1 variants. Allosteric integrase inhibitors (ALLINIs) are an emerging class of HIV-1 antagonists that are orthogonal to the current antiretroviral drugs. These small molecules act as highly specific molecular glue, which triggers the aggregation of HIV-1 integrase. In this work, we present high-resolution crystal structures that reveal the crucial interactions made by two potent ALLINIs, namely, BI-D and Pirmitegravir, with HIV-1 integrase. Our r
Lapidus S, Liu F, Casanovas-Massana A, et al., 2022, <i>Plasmodium</i> infection is associated with cross-reactive antibodies to carbohydrate epitopes on the SARS-CoV-2 Spike protein, SCIENTIFIC REPORTS, Vol: 12, ISSN: 2045-2322
van Vliet AR, Chiduza GN, Maslen SL, et al., 2022, ATG9A and ATG2A form a heteromeric complex essential for autophagosome formation, MOLECULAR CELL, Vol: 82, Pages: 4324-+, ISSN: 1097-2765
Jozwik IK, Li W, Zhang D-W, et al., 2022, B-to-A transition in target DNA during retroviral integration, NUCLEIC ACIDS RESEARCH, Vol: 50, Pages: 8898-8918, ISSN: 0305-1048
Seow J, Khan H, Rosa A, et al., 2022, A neutralizing epitope on the SD1 domain of SARS-CoV-2 spike targeted following infection and vaccination, CELL REPORTS, Vol: 40, ISSN: 2211-1247
Ng KW, Faulkner N, Finsterbusch K, et al., 2022, SARS-CoV-2 S2-targeted vaccination elicits broadly neutralizing antibodies, SCIENCE TRANSLATIONAL MEDICINE, Vol: 14, ISSN: 1946-6234
Ijaz S, Dicks S, Jegatheesan K, et al., 2022, Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: antibody dynamics and linkage to severity of COVID-19 in hospital inpatients, Journal of Infection, Vol: 85, Pages: 152-160, ISSN: 0163-4453
Ballandras-Colas A, Chivukula V, Gruszka DT, et al., 2022, Multivalent interactions essential for lentiviral integrase function, NATURE COMMUNICATIONS, Vol: 13
Khan M, Rosadas C, Katsanovskaja K, et al., 2022, Simple, sensitive, specific self-sampling assay secures SARS-CoV-2 antibody signals in sero-prevalence and post-vaccine studies, Scientific Reports, Vol: 12, ISSN: 2045-2322
At-home sampling is key to large scale seroprevalence studies. Dried blood spot (DBS) self-sampling removes the need for medical personnel for specimen collection but facilitates specimen referral to an appropriately accredited laboratory for accurate sample analysis. To establish a highly sensitive and specific antibody assay that would facilitate self-sampling for prevalence and vaccine-response studies. Paired sera and DBS eluates collected from 439 sero-positive, 382 sero-negative individuals and DBS from 34 vaccine recipients were assayed by capture ELISAs for IgG and IgM antibody to SARS-CoV-2. IgG and IgM combined on DBS eluates achieved a diagnostic sensitivity of 97.9% (95%CI 96.6 to 99.3) and a specificity of 99.2% (95% CI 98.4 to 100) compared to serum, displaying limits of detection equivalent to 23 and 10 WHO IU/ml, respectively. A strong correlation (r = 0.81) was observed between serum and DBS reactivities. Reactivity remained stable with samples deliberately rendered inadequate, (p = 0.234) and when samples were accidentally damaged or 'invalid'. All vaccine recipients were sero-positive. This assay provides a secure method for self-sampling by DBS with a sensitivity comparable to serum. The feasibility of DBS testing in sero-prevalence studies and in monitoring post-vaccine responses was confirmed, offering a robust and reliable tool for serological monitoring at a population level.
Rosadas C, Khan M, Parker E, et al., 2022, Detection and quantification of antibody to SARS CoV 2 receptor binding domain provides enhanced sensitivity, specificity and utility, Journal of Virological Methods, Vol: 302, ISSN: 0166-0934
Accurate and sensitive detection of antibody to SARS-CoV-2 remains an essential component of the pandemic response. Measuring antibody that predicts neutralising activity and the vaccine response is an absolute requirement for laboratory-based confirmatory and reference activity. The viral receptor binding domain (RBD) constitutes the prime target antigen for neutralising antibody. A double antigen binding assay (DABA), providing the most sensitive format has been exploited in a novel hybrid manner employing a solid-phase S1 preferentially presenting RBD, coupled with a labelled RBD conjugate, used in a two-step sequential assay for detection and measurement of antibody to RBD (anti-RBD). This class and species neutral assay showed a specificity of 100% on 825 pre COVID-19 samples and a potential sensitivity of 99.6% on 276 recovery samples, predicting quantitatively the presence of neutralising antibody determined by pseudo-type neutralisation and by plaque reduction. Anti-RBD is also measurable in ferrets immunised with ChadOx1 nCoV-19 vaccine and in humans immunised with both AstraZeneca and Pfizer vaccines. This assay detects anti-RBD at presentation with illness, demonstrates its elevation with disease severity, its sequel to asymptomatic infection and its persistence after the loss of antibody to the nucleoprotein (anti-NP). It also provides serological confirmation of prior infection and offers a secure measure for seroprevalence and studies of vaccine immunisation in human and animal populations. The hybrid DABA also displays the attributes necessary for the detection and quantification of anti-RBD to be used in clinical practice. An absence of detectable anti-RBD by this assay predicates the need for passive immune prophylaxis in at-risk patients.
Maertens GN, Engelman AN, Cherepanov P, 2022, Structure and function of retroviral integrase, Nature Reviews Microbiology, Vol: 20, Pages: 20-34, ISSN: 1740-1526
A hallmark of retroviral replication is establishment of the proviral state, wherein a DNA copy of the viral RNA genome is stably incorporated into a host cell chromosome. Integrase is the viral enzyme responsible for the catalytic steps involved in this process, and integrase strand transfer inhibitors are widely used to treat people living with HIV. Over the past decade, a series of X-ray crystallography and cryogenic electron microscopy studies have revealed the structural basis of retroviral DNA integration. A variable number of integrase molecules congregate on viral DNA ends to assemble a conserved intasome core machine that facilitates integration. The structures additionally informed on the modes of integrase inhibitor action and the means by which HIV acquires drug resistance. Recent years have witnessed the development of allosteric integrase inhibitors, a highly promising class of small molecules that antagonize viral morphogenesis. In this Review, we explore recent insights into the organization and mechanism of the retroviral integration machinery and highlight open questions as well as new directions in the field.
Davis C, Logan N, Tyson G, et al., 2021, Reduced neutralisation of the Delta (B.1.617.2) SARS-CoV-2 variant of concern following vaccination, PLOS PATHOGENS, Vol: 17, ISSN: 1553-7366
Arulkumaran N, Snow TAC, Kulkarni A, et al., 2021, Influence of IL-6 levels on patient survival in COVID-19, JOURNAL OF CRITICAL CARE, Vol: 66, Pages: 123-125, ISSN: 0883-9441
Alrubayyi A, Gea-Mallorqui E, Touizer E, et al., 2021, Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV, NATURE COMMUNICATIONS, Vol: 12
Barski MS, Vanzo T, Zhao XZ, et al., 2021, Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures (vol 12, 4996, 2021), NATURE COMMUNICATIONS, Vol: 12
Cherepanov P, 2021, The KT Jeang Retrovirology prize 2021: Peter Cherepanov, RETROVIROLOGY, Vol: 18
Deakin CT, Cornish GH, Ng KW, et al., 2021, Favorable antibody responses to human coronaviruses in children and adolescents with autoimmune rheumatic diseases, MED, Vol: 2, Pages: 1093-+, ISSN: 2666-6340
Maertens G, Barski MS, Vanzo T, et al., 2021, Structural basis for the inhibition of HTLV-1 integration inferred from cryo-EM deltaretroviral intasome structures, Nature Communications, Vol: 12, Pages: 1-10, ISSN: 2041-1723
Between 10 and 20 million people worldwide are infected with the human T-celllymphotropic virus type 1 (HTLV-1). Despite causing life-threateningpathologies there is no therapeutic regimen for this deltaretrovirus. Here, wescreened a library of integrase strand transfer inhibitor (INSTI) candidates builtaround several chemical scaffolds to determine their effectiveness in limitingHTLV-1 infection. Naphthyridines with substituents in position 6 emerged as themost potent compounds against HTLV-1, with XZ450 having highest efficacy invitro. Using single-particle cryo-electron microscopy we visualised XZ450 aswell as the clinical HIV-1 INSTIs raltegravir and bictegravir bound to the activesite of the deltaretroviral intasome. The structures reveal subtle differences inthe coordination environment of the Mg2+ ion pair involved in the interactionwith the INSTIs. Our results elucidate the binding of INSTIs to the HTLV-1intasome and support their use for pre-exposure prophylaxis and possiblyfuture treatment of HTLV-1 infection.
Arulkumaran N, Snow TAC, Kulkarni A, et al., 2021, Sex differences in immunological responses to COVID-19: a cross-sectional analysis of a single-centre cohort, BRITISH JOURNAL OF ANAESTHESIA, Vol: 127, Pages: E75-E78, ISSN: 0007-0912
Arulkumaran N, Snow TAC, Kulkarni A, et al., 2021, Defining Potential Therapeutic Targets in Coronavirus Disease 2019: A Cross-Sectional Analysis of a Single-Center Cohort., Crit Care Explor, Vol: 3
OBJECTIVES: Multiple mechanisms have been proposed to explain disease severity in coronavirus disease 2019. Therapeutic approaches need to be underpinned by sound biological rationale. We evaluated whether serum levels of a range of proposed coronavirus disease 2019 therapeutic targets discriminated between patients with mild or severe disease. DESIGN: A search of ClinicalTrials.gov identified coronavirus disease 2019 immunological drug targets. We subsequently conducted a retrospective observational cohort study investigating the association of serum biomarkers within the first 5 days of hospital admission relating to putative therapeutic biomarkers with illness severity and outcome. SETTING: University College London, a tertiary academic medical center in the United Kingdom. PATIENTS: Patients admitted to hospital with a diagnosis of coronavirus disease 2019. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Eighty-six patients were recruited, 44 (51%) with mild disease and 42 (49%) with severe disease. We measured levels of 10 cytokines/signaling proteins related to the most common therapeutic targets (granulocyte-macrophage colony-stimulating factor, interferon-α2a, interferon-β, interferon-γ, interleukin-1β, interleukin-1 receptor antagonist, interleukin-6, interleukin-7, interleukin-8, tumor necrosis factor-α), immunoglobulin G antibodies directed against either coronavirus disease 2019 spike protein or nucleocapsid protein, and neutralization titers of antibodies. Four-hundred seventy-seven randomized trials, including 168 different therapies against 83 different pathways, were identified. Six of the 10 markers (interleukin-6, interleukin-7, interleukin-8, interferon-α2a, interferon-β, interleukin-1 receptor antagonist) discriminated between patients with mild and severe disease, although most were similar or only modestly raised above that seen in healthy volunteers. A similar proportion of patients with mild or sever
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