scholarly journals Global assessment ofMycobacterium aviumsubspecieshominissuisgenetic requirement for growth and virulence

2019 ◽  
Author(s):  
Marte S. Dragset ◽  
Thomas R. Ioerger ◽  
Maja Loevenich ◽  
Markus Haug ◽  
Niruja Sivakumar ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Gene Function ◽  
Drug Targets ◽  
Virulence Genes ◽  
Genetic Manipulation ◽  
Specific Treatment ◽  
Hypothetical Protein ◽  
Transposon Mutagenesis ◽  
High Density

AbstractNontuberculous mycobacterial infections caused by the opportunistic pathogenMycobacterium aviumsubsp.hominissuis(MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence andin vitrogrowth, and organized ~3500 identified transposon mutants for hypothesis-driven research. The majority (71 %) of the genes we identified as essential for MAHin vitrohad a growth-essential mutual ortholog in the related and highly virulentM. tuberculosis(Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. Strikingly, > 97 % of the MAH virulence genes had a mutual ortholog inMtb. Two of the three virulence genes specific to MAH (i.e. noMtbmutual orthologs) were PPE proteins, a family of proteins unique to mycobacteria and highly associated with virulence. Finally, we validated novel genes as required for successful MAH infection; one encoding a probable MFS transporter and another a hypothetical protein located in immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection.Author summaryPulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by theM. aviumcomplex (MAC), whereof >90% arise fromMycobacterium aviumsubsp.hominissuis(MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention and diagnostics, we need to understand the underlying mechanisms of infection. While genetic manipulation of pathogens is crucial to study pathogenesis,M. avium(Mav) has been found notoriously hard to engineer. Here, we identify an MAH strain highly susceptible to high-density transposon mutagenesis and transformation, facilitating genetic engineering and analysis of gene function. We provide crucial insights into this strain’s global genetic requirements for growth and infection. Surprisingly, we find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogenM. tuberculosis(Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment, or translate to broader treatment of related mycobacterial diseases.

scholarly journals Global Assessment of Mycobacterium avium subsp. hominissuis Genetic Requirement for Growth and Virulence

mSystems ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Marte S. Dragset ◽  
Thomas R. Ioerger ◽  
Maja Loevenich ◽  
Markus Haug ◽  
Niruja Sivakumar ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Drug Targets ◽  
Virulence Genes ◽  
Genetic Manipulation ◽  
Specific Treatment ◽  
Opportunistic Pathogen ◽  
Hypothetical Protein ◽  
Major Facilitator Superfamily ◽  
Content Type

ABSTRACT Nontuberculous mycobacterial infections caused by the opportunistic pathogen Mycobacterium avium subsp. hominissuis (MAH) are currently receiving renewed attention due to increased incidence combined with difficult treatment. Insights into the disease-causing mechanisms of this species have been hampered by difficulties in genetic manipulation of the bacteria. Here, we identified and sequenced a highly transformable, virulent MAH clinical isolate susceptible to high-density transposon mutagenesis, facilitating global gene disruption and subsequent investigation of MAH gene function. By transposon insertion sequencing (TnSeq) of this strain, we defined the MAH genome-wide genetic requirement for virulence and in vitro growth and organized ∼3,500 identified transposon mutants for hypothesis-driven research. The majority (96%) of the genes we identified as essential for MAH in vitro had a mutual ortholog in the related and highly virulent Mycobacterium tuberculosis (Mtb). However, passaging our library through a mouse model of infection revealed a substantial number (54% of total hits) of novel virulence genes. More than 97% of the MAH virulence genes had a mutual ortholog in Mtb. Finally, we validated novel genes required for successful MAH infection: one encoding a probable major facilitator superfamily (MFS) transporter and another encoding a hypothetical protein located in the immediate vicinity of six other identified virulence genes. In summary, we provide new, fundamental insights into the underlying genetic requirement of MAH for growth and host infection. IMPORTANCE Pulmonary disease caused by nontuberculous mycobacteria is increasing worldwide. The majority of these infections are caused by the Mycobacterium avium complex (MAC), whereof >90% are due to Mycobacterium avium subsp. hominissuis (MAH). Treatment of MAH infections is currently difficult, with a combination of antibiotics given for at least 12 months. To control MAH by improved therapy, prevention, and diagnostics, we need to understand the underlying mechanisms of infection. Here, we provide crucial insights into MAH’s global genetic requirements for growth and infection. We find that the vast majority of genes required for MAH growth and virulence (96% and 97%, respectively) have mutual orthologs in the tuberculosis-causing pathogen M. tuberculosis (Mtb). However, we also find growth and virulence genes specific to MAC species. Finally, we validate novel mycobacterial virulence factors that might serve as future drug targets for MAH-specific treatment or translate to broader treatment of related mycobacterial diseases.

Discerning novel drug targets for treating Mycobacterium avium ss. paratuberculosis-associated autoimmune disorders: an in silico approach

2020 ◽  
Author(s):  
Anjali Garg ◽  
Neelja Singhal ◽  
Manish Kumar
Keyword(s):  
Multiple Sclerosis ◽  
Autoimmune Diseases ◽  
Mycobacterium Avium ◽  
In Silico ◽  
Drug Targets ◽  
Molecular Mimicry ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
In Silico Analysis ◽  
Experimental Drugs

Abstract Mycobacterium avium subspecies paratuberculosis (MAP) exhibits ‘molecular mimicry’ with the human host resulting in several autoimmune diseases such as multiple sclerosis, type 1 diabetes mellitus (T1DM), Hashimoto’s thyroiditis, Crohn’s disease (CD), etc. The conventional therapy for autoimmune diseases includes immunosuppressants or immunomodulators that treat the symptoms rather than the etiology and/or causative mechanism(s). Eliminating MAP–the etiopathological agent might be a better strategy to treat MAP-associated autoimmune diseases. In this case study, we conducted a systematic in silico analysis to identify the metabolic chokepoints of MAP’s mimicry proteins and their interacting partners. The probable inhibitors of chokepoint proteins were identified using DrugBank. DrugBank molecules were stringently screened and molecular interactions were analyzed by molecular docking and ‘off-target’ binding. Thus, we identified 18 metabolic chokepoints of MAP mimicry proteins and 13 DrugBank molecules that could inhibit three chokepoint proteins viz. katG, rpoB and narH. On the basis of molecular interaction between drug and target proteins finally eight DrugBank molecules, viz. DB00609, DB00951, DB00615, DB01220, DB08638, DB08226, DB08266 and DB07349 were selected and are proposed for treatment of three MAP-associated autoimmune diseases namely, T1DM, CD and multiple sclerosis. Because these molecules are either approved by the Food and Drug Administration or these are experimental drugs that can be easily incorporated in clinical studies or tested in vitro. The proposed strategy may be used to repurpose drugs to treat autoimmune diseases induced by other pathogens.

scholarly journals The Mycobacterium avium subsp. paratuberculosis MAP3464 Gene Encodes an Oxidoreductase Involved in Invasion of Bovine Epithelial Cells through the Activation of Host Cell Cdc42

2007 ◽  
Vol 76 (1) ◽  
pp. 170-178 ◽  
Author(s):  
Marta Alonso-Hearn ◽  
Dilip Patel ◽  
Lia Danelishvili ◽  
Lisbeth Meunier-Goddik ◽  
Luiz E. Bermudez
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Mycobacterium Avium ◽  
Hypothetical Protein ◽  
Complete Restoration ◽  
Mdbk Cells ◽  
Transposon Mutants ◽  
Infected Cells ◽  
Mycobacterium Avium Subsp Paratuberculosis

ABSTRACT Mycobacterium avium subsp. paratuberculosis infection of cattle takes place through the intestinal mucosa. To identify M. avium subsp. paratuberculosis genes associated with the invasion of bovine epithelial cells in vitro, we screened a library of transposon mutants. Several mutants of M. avium subsp. paratuberculosis were identified which invaded Madin-Darby bovine kidney (MDBK) epithelial cells less efficiently than wild-type (wt) M. avium subsp. paratuberculosis. The ΔOx mutant had the transposon located in the MAP3464 gene, a putative oxidoreductase gene whose expression is upregulated upon bacterial contact with MDBK cells. Complete restoration of invasion comparable to that for the wt bacterium was achieved by introducing a copy of the complete oxidoreductase operon into the ΔOx mutant. Immunoprecipitation and Western blot analysis indicated that wt M. avium subsp. paratuberculosis activates Cdc42 and RhoA pathways of internalization 15 and 60 min after infection of the host cell, respectively. The ΔOx mutant, however, failed to activate the Cdc42 pathway. To determine whether an M. avium subsp. paratuberculosis protein delivered to the host cell mediates the entry of the wt bacterium by activation of the Cdc42 pathway, affinity precipitation of active Cdc42 from MDBK-infected cells followed by mass spectrometry was carried out. We identified a 17-amino-acid bacterial peptide associated with the Cdc42 of cells infected with wt M. avium subsp. paratuberculosis but not with the ΔOx mutant. The sequence of the peptide matches MAP3985c, a hypothetical protein, possibly functioning as a putative Cdc42 effector. These findings reveal a novel signaling pathway activated during M. avium subsp. paratuberculosis entry that links the product of MAP3464 gene to activation of Cdc42 in the host cell.

scholarly journals Gene Function Analysis in the Ubiquitous Human Commensal and PathogenMalasseziaGenus

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Giuseppe Ianiri ◽  
Anna F. Averette ◽  
Joanne M. Kingsbury ◽  
Joseph Heitman ◽  
Alexander Idnurm
Keyword(s):  
Gene Function ◽  
Genetic Manipulation ◽  
Function Analysis ◽  
Exogenous Dna ◽  
Melanin Biosynthesis ◽  
Disease Symptoms ◽  
Wide Range ◽  
Transformation Methods ◽  
Gene Function Analysis

ABSTRACTThe genusMalasseziaincludes 14 species that are found on the skin of humans and animals and are associated with a number of diseases. Recent genome sequencing projects have defined the gene content of all 14 species; however, to date, genetic manipulation has not been possible for any species within this genus. Here, we develop and then optimize molecular tools for the transformation ofMalassezia furfurandMalassezia sympodialisusingAgrobacterium tumefaciensdelivery of transfer DNA (T-DNA) molecules. These T-DNAs can insert randomly into the genome. In the case ofM. furfur, targeted gene replacements were also achieved via homologous recombination, enabling deletion of theADE2gene for purine biosynthesis and of theLAC2gene predicted to be involved in melanin biosynthesis. Hence, the introduction of exogenous DNA and direct gene manipulation are feasible inMalasseziaspecies.IMPORTANCESpecies in the genusMalasseziaare a defining component of the microbiome of the surface of mammals. They are also associated with a wide range of skin disease symptoms. Many species are difficult to culturein vitro, and although genome sequences are available for the species in this genus, it has not been possible to assess gene function to date. In this study, we pursued a series of possible transformation methods and identified one that allows the introduction of DNA into two species ofMalassezia, including the ability to make targeted integrations into the genome such that genes can be deleted. This research opens a new direction in terms of now being able to analyze gene functions in this little understood genus. These tools will contribute to define the mechanisms that lead to the commensalism and pathogenicity in this group of obligate fungi that are predominant on the skin of mammals.

scholarly journals Characterization and Pathogenic Significance of Vibrio vulnificus Antigens Preferentially Expressed in Septicemic Patients

2003 ◽  
Vol 71 (10) ◽  
pp. 5461-5471 ◽  
Author(s):  
Young Ran Kim ◽  
Shee Eun Lee ◽  
Choon Mee Kim ◽  
Soo Young Kim ◽  
Eun Kyoung Shin ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Pathogenic Bacteria ◽  
Virulence Genes ◽  
Vibrio Vulnificus ◽  
Lethal Dose ◽  
Hypothetical Protein ◽  
Expression Library

ABSTRACT Many important virulence genes of pathogenic bacteria are preferentially expressed in vivo. We used the recently developed in vivo-induced antigen technology (IVIAT) to identify Vibrio vulnificus genes induced in vivo. An expression library of V. vulnificus was screened by colony blot analysis by using pooled convalescent-phase serum that had been thoroughly adsorbed with in vitro-expressed V. vulnificus whole cells and lysates. Twelve clones were selected, and the sequences of the insert DNAs were analyzed. The DNA sequences showed homologies with genes encoding proteins of diverse functions: these functions included chemotaxis (a methyl-accepting chemotaxis protein), signaling (a GGDEF-containing protein and a putative serine/threonine kinase), biosynthesis and metabolism (PyrH, PurH, and IlvC), secretion (TatB and plasmid Achromobacter secretion [PAS] factor), transcriptional activation (IlvY and HlyU), and the activity of a putative lipoprotein (YaeC). In addition, one identified open reading frame encoded a hypothetical protein. Isogenic mutants of the 12 in vivo-expressed (ive) genes were constructed and tested for cytotoxicity. Cytotoxic activity of the mutant strains, as measured by lactate dehydrogenase release from HeLa cells, was nearly abolished in pyrH, purH, and hlyU mutants. The intraperitoneal 50% lethal dose in mice increased by ca. 10- to 50-fold in these three mutants. PyrH and PurH seem to be essential for in vivo growth. HlyU appears to be one of the master regulators of in vivo virulence expression. The successful identification of ive genes responsible for the in vivo bacterial virulence, as done in the present study, demonstrates the usefulness of IVIAT for the detection of new virulence genes.

scholarly journals Chromosome-Wide Analysis of Gene Function by RNA Interference in the African Trypanosome

2006 ◽  
Vol 5 (9) ◽  
pp. 1539-1549 ◽  
Author(s):  
Chandra Subramaniam ◽  
Paul Veazey ◽  
Seth Redmond ◽  
Jamie Hayes-Sinclair ◽  
Emma Chambers ◽  
...  
Keyword(s):  
Rna Interference ◽  
Gene Function ◽  
Drug Targets ◽  
Cell Cycle Progression ◽  
In Silico Analysis ◽  
Phenotypic Analysis ◽  
Online Data ◽  
Systematic Analysis ◽  
Chromosome I

ABSTRACT Trypanosomatids of the order Kinetoplastida are major contributors to global disease and morbidity, and understanding their basic biology coupled with the development of new drug targets represents a critical need. Additionally, trypanosomes are among the more accessible divergent eukaryote experimental systems. The genome of Trypanosoma brucei contains 8,131 predicted open reading frames (ORFs), of which over half have no known homologues beyond the Kinetoplastida and a substantial number of others are poorly defined by in silico analysis. Thus, a major challenge following completion of the T. brucei genome sequence is to obtain functional data for all trypanosome ORFs. As T. brucei is more experimentally tractable than the related Trypanosoma cruzi and Leishmania spp. and shares >75% of their genes, functional analysis of T. brucei has the potential to inform a range of parasite biology. Here, we report methods for systematic mRNA ablation by RNA interference (RNAi) and for phenotypic analysis, together with online data dissemination. This represents the first systematic analysis of gene function in a parasitic organism. In total, 210 genes have been targeted in the bloodstream form parasite, representing an essentially complete phenotypic catalogue of chromosome I together with a validation set. Over 30% of the chromosome I genes generated a phenotype when targeted by RNAi; most commonly, this affected cell growth, viability, and/or cell cycle progression. RNAi against approximately 12% of ORFs was lethal, and an additional 11% had growth defects but retained short-term viability in culture. Although we found no evidence for clustering or a bias towards widely evolutionarily conserved genes within the essential ORF cohort, the putative chromosome I centromere is adjacent to a domain containing genes with no associated phenotype. Involvement of such a large proportion of genes in robust growth in vitro indicates that a high proportion of the expressed trypanosome genome is required for efficient propagation; many of these gene products represent potential drug targets.

scholarly journals Characterization and Application of a Glucose-Repressible Promoter in Francisella tularensis

2008 ◽  
Vol 74 (7) ◽  
pp. 2161-2170 ◽  
Author(s):  
Joseph Horzempa ◽  
Deanna M. Tarwacki ◽  
Paul E. Carlson ◽  
Cory M. Robinson ◽  
Gerard J. Nau
Keyword(s):  
Gene Expression ◽  
Francisella Tularensis ◽  
Gene Function ◽  
Fluorescent Protein ◽  
Genomic Sequence ◽  
Protein A ◽  
Hypothetical Protein ◽  
Protein Levels

ABSTRACT Francisella tularensis, the causative agent of tularemia, is a category A biodefense agent. The examination of gene function in this organism is limited due to the lack of available controllable promoters. Here, we identify a promoter element of F. tularensis LVS that is repressed by glucose (termed the Francisella glucose-repressible promoter, or FGRp), allowing the management of downstream gene expression. In bacteria cultured in medium lacking glucose, this promoter induced the expression of a red fluorescent protein allele, tdtomato. FGRp activity was used to produce antisense RNA of iglC, an important virulence factor, which severely reduced IglC protein levels. Cultivation in glucose-containing medium restored IglC levels, indicating the usefulness of this promoter for controlling both exogenous and chromosomal gene expression. Moreover, FGRp was shown to be active during the infection of human macrophages by using the fluorescence reporter. In this environment, the FGRp-mediated expression of antisense iglC by F. tularensis LVS resulted in reduced bacterial fitness, demonstrating the applicability of this promoter. An analysis of the genomic sequence indicated that this promoter region controls a gene, FTL_0580, encoding a hypothetical protein. A deletion analysis determined the critical sites essential for FGRp activity to be located within a 44-bp region. This is the first report of a conditional promoter and the use of antisense constructs in F. tularensis, valuable genetic tools for studying gene function both in vitro and in vivo.

scholarly journals Identifying the essential genes of Mycobacterium avium subsp. hominissuis with Tn-Seq using a rank-based filter procedure

2019 ◽  
Author(s):  
William M. Matern ◽  
Robert L. Jenquin ◽  
Joel S. Bader ◽  
Petros C. Karakousis
Keyword(s):  
Mycobacterium Avium ◽  
Genetic Manipulation ◽  
Laboratory Strain ◽  
New Drugs ◽  
Mycobacterium Avium Subsp ◽  
Growth Defect ◽  
Future Research ◽  
Computational Technique ◽  
Curative Therapy

AbstractMycobacterium avium (Mav) is increasingly recognized as a significant cause of morbidity, particularly in elderly patients or those with immune deficiency or underlying structural lung disease. Generally, Mav infection is treated with 2-3 antimicrobial drugs for at least 12 months. Identification of genes essential for Mav growth may yield novel strategies for improving curative therapy. We have generated saturating genome-wide transposon mutant pools in a commonly used laboratory strain of Mycobacterium avium subsp. hominissuis (MAC109) and developed a computational technique for classifying annotated genomic features as essential (ES), growth defect (GD), growth advantage (GA), or no-effect (NE) based on the in vitro effect of disruption by transposon. We identified 270 features as ES with 230 of these overlapping with ES features in Mycobacterium tuberculosis. These results may be useful for identifying drug targets or for informing studies requiring genetic manipulation of Mycobacterium avium, which should seek to avoid disrupting ES features to ensure bacterial viability.ImportanceMycobacterium avium subsp. hominissuis is an emerging cause of morbidity in vulnerable populations in many countries. It is known to be particularly difficult to treat, often requiring years of antibiotic therapy. In this study we report the genes of Mycobacterium avium subsp. hominissuis that are required for the organism to grow in vitro. Our findings may help guide future research into identifying new drugs to improve the treatment of this serious infection.

scholarly journals Development of Transposon Mutagenesis for Chlamydia muridarum

2019 ◽  
Vol 201 (23) ◽  
Author(s):  
Yibing Wang ◽  
Scott D. LaBrie ◽  
Steven J. Carrell ◽  
Robert J. Suchland ◽  
Zoe E. Dimond ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Transposon Mutagenesis ◽  
Content Type ◽  
Chlamydia Muridarum ◽  
Transposon Mutants ◽  
Transposon Insertions ◽  
Glycogen Biosynthesis

ABSTRACT Functional genetic analysis of Chlamydia has been a challenge due to the historical genetic intractability of Chlamydia, although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for Chlamydia muridarum, a mouse-adapted Chlamydia species that is widely used in Chlamydia infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing C. muridarum transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the C. muridarum chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in glgB, pmpI, pmpA, and pmpD were investigated further for in vitro and in vivo phenotypes, including growth, inclusion morphology, and attachment to host cells. The glgB mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 pmp mutants, pmpI was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for C. muridarum. The generation of a complete library of C. muridarum mutants will ultimately enable comprehensive identification of the functional genetic requirements for Chlamydia infection in vivo. IMPORTANCE Historical issues with genetic manipulation of Chlamydia have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for C. muridarum, a mouse-adapted Chlamydia species that is widely used for in vivo investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for C. trachomatis. We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized in vitro phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.

The Natural Course of Defibrination Syndrome Caused by Echis Colorata Venom in Man

1974 ◽  
Vol 31 (03) ◽  
pp. 420-428 ◽  
Author(s):  
M Fainaru ◽  
S Eisenberg ◽  
N Manny ◽  
C Hershko
Keyword(s):  
Factor Viii ◽  
Blood Coagulation ◽  
Major Bleeding ◽  
Renal Damage ◽  
Specific Treatment ◽  
Natural Course ◽  
Factor V ◽  
Thrombocyte Count

SummaryThe natural course of defibrination syndrome caused by Echis colorata venom (ECV) in five patients is reported. All patients developed afibrinogenemia within six hours after the bite. Concomitantly a depression in factor V was recorded. Factor VIII and thrombocyte count in blood were normal in most patients. In the light of the known effects of ECV on blood coagulation in vivo and in vitro it is concluded that the afibrinogenemia is due to intravascular clotting.Four patients had transient renal damage, manifested by oliguria, azotemia, albuminuria and cylindruria, ascribed to microthrombi in the renal glomeruli.After the bite, the natural course was benign, no major bleeding was observed, and all signs of coagulopathy reverted to normal within 7 days. Therefore we recommend no specific treatment for this condition. In the case of heavily bleeding patients, administration of antiserum against ECV and/or heparin should be considered.

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