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See detailStudies of the Domains II and III of Bacillus subtilis PBP4a in relation with the protein localization
Vanden Broeck, Arnaud ULg; Van Der Heiden, Edwige ULg; Sauvage, Eric ULg et al

Poster (2014, April 23)

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been ... [more ▼]

Bacillus subtilis PBP4a belongs to the class-C1 PBPs characterized by two internal additional domains of unknown function. Seven lysine residues (K) are protruding from domain II. Four of them have been mutated in glutamine residues (Q). Both proteins (WT and Mut4KQ PBP4a) have been produced without signal peptide in E. coli and their sub-cellular localizations determined by measuring the DD-carboxypeptidase activities in the different compartments (cytoplasmic vs membrane attached proteins). In order to detect a possible influence of the PBP4a domain III in the localization of the protein, its encoding sequence has been cloned into pET-28b-BlaP, a vector allowing the production of WT BlaP β-lactamase or BlaP/DIII chimeric protein (with domain III inserted in a permissive loop of BlaP). The nitrocefin hydrolysis activities of BlaP or BlaP/DIII have been measured in the different cellular compartments. [less ▲]

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See detailStructure-function relationship of oligomers in amyloidoses
Huynen, Céline ULg

Scientific conference (2014, March 20)

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See detailSwitching to the Dark Side: Repositioning of Polyglutamine Repeat Promotes Amyloid Fibril Formation by the Model Protein, β-Lactamase BlaP
Thorn, David ULg; Pain, Coralie ULg; Scarafone, Natacha et al

Conference (2013, November 20)

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within ... [more ▼]

Background: The expansion of polyglutamine (polyQ) repeats is associated with an increased propensity of the protein to aggregate into amyloid fibrils. There are ten human proteins presently known within which polyQ expansion above a threshold length, e.g. 35-50 residues, leads to ten distinct neurodegenerative disorders [1], the most well-known being Huntington’s disease. While repeat length, aggregation, and disease are well correlated, recent studies suggest the non-polyQ regions of these proteins can also play a significant role, both preventative and facilitative, in the aggregation process. With the aim of exploring this role in more detail, we have engineered chimeric proteins via the insertion of polyQ repeats of various length (23, 30, 55, 79 Q) into two sites of antibiotic resistance enzyme BlaP β-lactamase from Bacillus licheniformis 749/C [2]. Questions addressed: How does polyQ repeat position affect the structure, stability and aggregation of polyQ proteins? Methods: Aggregation kinetics determined by monitoring the decrease in soluble protein fraction over time. Aggregate morphology examined by transmission electron microscopy. Protein stability derived from thermal or chemical unfolding transitions monitored by far-UV CD and intrinsic fluorescence. Results and discussion: PolyQ insertion at either of the two positions led to a decrease in thermodynamic stability that was largely independent of polyQ length. Chimeras with polyQ insertions at position 216 were destabilised to a much greater extent than those with insertions at position 197. The reduced stability of the 216 chimeras was associated with an increased aggregation propensity: (i) the minimum polyQ length leading to aggregation was lower, and (ii) the aggregation rate was significantly higher than that observed by 197 chimeras with equivalent polyQ lengths. Remarkably, the two sites of polyQ insertion are indeed very similar, both residing within flexible loop regions between stable α-helices. Moreover, the 216 chimeras exhibited a higher aggregation propensity than their 197 counterparts even under denaturing conditions, suggesting the disparity between the two chimeras cannot be accounted for by structural differences alone. These findings highlight the strong and complex influence of the overall protein context on polyQ-mediated aggregation. The molecular basis for the observed changes in stability and aggregation propensity is the subject of on-going work. [less ▲]

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See detailRole of non-polyQ regions on the different steps of amyloid fibril formation by polyQ proteins
Huynen, Céline ULg

Poster (2013, November 15)

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a ... [more ▼]

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils. [less ▲]

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Peer Reviewed
See detailClass A β -Lactamases as Versatile Scaffolds to Create Hybrid Enzymes: Applications from Basic Research to Medicine
Huynen, Céline ULg; Filée, Patrice; Matagne, André ULg et al

in BioMed Research International (2013), 2013

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽 ... [more ▼]

Designing hybrid proteins is a major aspect of protein engineering and covers a very wide range of applications frombasic research to medical applications. This review focuses on the use of class A 𝛽-lactamases as versatile scaffolds to design hybrid enzymes (referred to as 𝛽-lactamase hybrid proteins, BHPs) in which an exogenous peptide, protein or fragment thereof is inserted at various permissive positions.We discuss how BHPs can be specifically designed to create bifunctional proteins, to produce and to characterize proteins that are otherwise difficult to express, to determine the epitope of specific antibodies, to generate antibodies against nonimmunogenic epitopes, and to better understand the structure/function relationship of proteins. [less ▲]

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See detailRole of non-polyQ regions on the aggregation of polyQ proteins into amyloid fibrils triggered by polyQ expansions
Huynen, Céline ULg

Poster (2013, July 22)

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a ... [more ▼]

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils. Finally, they clearly indicate that the oligomers of BlaP197(Gln)79 observed at the early stage of the aggregation process are on the pathway of amyloid fibril formation, and likely constitute the aggregation nucleus. [less ▲]

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See detailRole of non-polyQ regions on the aggregation of polyQ proteins into amyloid fibrils triggered by polyQ expansions
Huynen, Céline ULg

Conference (2013, July 21)

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a ... [more ▼]

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils. Finally, they clearly indicate that the oligomers of BlaP197(Gln)79 observed at the early stage of the aggregation process are on the pathway of amyloid fibril formation, and likely constitute the aggregation nucleus. [less ▲]

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See detailInfluence of the protein context in the aggregation of polyQ proteins into amyloid fibrils
Huynen, Céline ULg

Poster (2013, April 18)

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a ... [more ▼]

Ten neurodegenerative diseases, referred to as polyglutamine (polyQ) diseases, are associated with the aggregation into amyloid fibrils of ten different proteins containing a polyQ expansion higher than a pathological threshold comprised between 35 to 45Q (1, 2). A large body of evidence indicate that the polyQ expansion is the critical determinant for the aggregation of these polyQ proteins. The aggregation process of polyQ proteins is, however, still not well understood. To better understand this mechanism at a molecular level, we have characterized model polyQ proteins made of the β-lactamase BlaP from Bacillus licheniformis 749/C and a polyQ tract of 0 to 79Q inserted either at position 197 or position 216 of BlaP. Those chimeras recapitulate the same aggregation behaviours than that of disease-associated polyQ proteins: there is a glutamine threshold for the aggregation into amyloid fibrils and the anticipation phenomenon. Most importantly, the threshold critically depends on the structural integrity of BlaP (3) which would impose some conformational and/or sterical constraints to the polyQ tract. Moreover the position of the polyQ insertion into BlaP modifies the aggregation propensity of BlaP chimeras. The present work aims to further investigate (i) how the protein context affects the different phases of the aggregation phenomenon (i.e. the nucleation and elongation phases) and (ii) the role of the oligomers formed during the early time of the aggregation process. The techniques used are mainly (1) quartz crystal microbalance (QCM) and atomic force microscopy (AFM) to study the elongation step of amyloid fibril formation and (2) dynamic light scattering (DLS) to study the evolution of the different populations formed during the aggregation time course. The results of these experiments indicate that the native conformation of BlaP197(Gln)55 interferes mainly with the nucleation but not with the elongation step of amyloid fibril formation. Moreover, these results demonstrate that the sequences flanking the polyQ tract significantly influence its propensity to elongate amyloid fibrils. Finally, they clearly indicate that the oligomers of BlaP197(Gln)79 observed at the early stage of the aggregation process are on the pathway of amyloid fibril formation, and likely constitute the aggregation nucleus. [less ▲]

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See detailInfluence of the protein context on the propensity of polyglutamine tracts to trigger amyloid fibril formation
Huynen, Céline ULg

Conference (2012, November 16)

10 neurodegenerative diseases are associated with 10 proteins containing an expanded polyglutamine (polyQ) tract higher than a threshold of 35-45Q. This polyQ tracts trigger the aggregation of the ... [more ▼]

10 neurodegenerative diseases are associated with 10 proteins containing an expanded polyglutamine (polyQ) tract higher than a threshold of 35-45Q. This polyQ tracts trigger the aggregation of the proteins into amyloid fibrils that could play an important role in the disease. It is therefore necessary to investigate the aggregation properties of polyQ proteins. Accordingly, we have created model proteins made of the β-lactamase BlaP with 23 to 79Q inserted at positions 197 or 216. Their aggregation behaviour recapitulate those of disease-associated proteins (i.e. a threshold and the anticipation phenomenon). Moreover, the threshold depends on the structural integrity of BlaP (which applies constraints to the polyQ). In this work, we investigate the role of the protein context in the aggregation process using two techniques: QCM-D coupled with AFM. We observe that the native conformation of BlaP is the barrier for fibril formation, interfering with the nucleation step. Moreover, the protein environment of the polyQ (position 197 or 216) influences its propensity to form fibrils. [less ▲]

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See detailInfluence of the protéin context on the propensity of polyglutamine tracts to trigger protein aggregation into amyloid fibrils
Huynen, Céline ULg

Conference (2012, September 13)

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of ... [more ▼]

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathological tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally large and relatively insoluble, they are difficult to produce and handle experimentally. We have therefore decided to study the aggregation properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted either at positions 197 or 216 of BlaP. The aggregation behaviour of BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (i.e. a threshold) required for the chimeras’ aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and of oligomers on the aggregation process of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). QCM-D studies indicate that, under native condition, there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras containing a polyQ tract in position 197. This threshold is lower than that observed for the aggregation assays monitored in solution in the absence of seed; it corresponds however to the threshold for fibril formation observed under denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. BlaP chimeras containing 55Q or more in position 216 of BlaP also elongate BlaP197(Gln)79 fibrils. Interestingly this rate of cross-elongation is faster than the elongation rate observed with BlaP197(Gln)55 and BlaP197(Gln)79. This observation demonstrates that the protein environment of the polyQ tract influences its propensity to form amyloid fibrils. Finally, preliminary DLS experiments indicate that oligomers formed by BlaP197(Gln)79 under native condition are on-pathway of amyloid fibril formation and could act as aggregation nuclei. [less ▲]

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See detailInfluence of protein context on the propensity of polyglutamine tracts to trigger protein aggregation into amyloid fibrils
Huynen, Céline ULg

Poster (2012, September 10)

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of ... [more ▼]

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathological tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally large and relatively insoluble, they are difficult to produce and handle experimentally. We have therefore decided to study the aggregation properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted either at positions 197 or 216 of BlaP. The aggregation behaviour of BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (i.e. a threshold) required for the chimeras’ aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and of oligomers on the aggregation process of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). QCM-D studies indicate that, under native condition, there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras containing a polyQ tract in position 197. This threshold is lower than that observed for the aggregation assays monitored in solution in the absence of seed; it corresponds however to the threshold for fibril formation observed under denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. BlaP chimeras containing 55Q or more in position 216 of BlaP also elongate BlaP197(Gln)79 fibrils. Interestingly this rate of cross-elongation is faster than the elongation rate observed with BlaP197(Gln)55 and BlaP197(Gln)79. This observation demonstrates that the protein environment of the polyQ tract influences its propensity to form amyloid fibrils. Finally, preliminary DLS experiments indicate that oligomers formed by BlaP197(Gln)79 under native condition are on-pathway of amyloid fibril formation and could act as aggregation nuclei. [less ▲]

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See detailInfluence of protein context on the propensity of polyglutamine tracts to induce protein aggregation into amyloid fibrils
Huynen, Céline ULg

Poster (2012, April 18)

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of ... [more ▼]

Nine neurodegenerative diseases, referred to as polyglutamine diseases, are associated with nine proteins containing an expanded polyglutamine (polyQ) tract. PolyQ tracts are encoded by a repetition of the CAG codon in the corresponding genes, and are present in proteins of healthy people. They are however pathogenic when their length, due to mutations, becomes higher than a threshold generally comprised between 35 and 45Q. Such pathogenic tracts trigger the aggregation of the proteins into amyloid-like aggregates that could play an important role in the disease. It is therefore necessary to investigate at a molecular level the aggregation process of polyQ proteins. Since proteins associated with polyQ diseases are generally big and relatively insoluble, they are difficult to produce and manipulate. We have therefore decided to study the aggregating properties of polyQ proteins by designing and characterizing model proteins made of a well-characterized host protein, the β-lactamase BlaP, and polyQ tracts of different lengths (23 to 79Q) inserted at position 197 of BlaP. The aggregating properties of these BlaP chimeras recapitulate those of proteins associated with polyQ diseases. We indeed observed that there is a minimal number of glutamines (threshold) required for chimeras aggregation into amyloid-like fibrils and that the kinetics of aggregation are faster with longer glutamine repeats. Most importantly, the value of the threshold for amyloid-like fibril formation seems to critically depend on the structural integrity of BlaP and thus on the constraints applied to the polyQ tract. In the present work, we investigate more deeply the role of the protein context and the role of oligomers in the process of aggregation of BlaP chimeras. This study is mainly based on the use of two techniques: the quartz crystal microbalance with dissipation (QCM-D) in combination with atomic force microscopy (AFM), and the dynamic light scattering (DLS). Preliminary QCM-D results indicate that in native condition there is a minimal number of glutamines required for the elongation of BlaP197(Gln)79 fibrils by BlaP chimeras. This threshold is different to that observed for the aggregation monitored in solution in absence of seed. Most interestingly it corresponds to the threshold observed in denaturing conditions. These observations suggest that the conformation of BlaP is the limiting step for amyloid fibril formation by interfering with the nucleation step. Preliminary DLS experiments indicate that the oligomers formed by BlaP197(Gln)79 in native condition are on the pathway of amyloid fibril formation and could act as aggregation nuclei. [less ▲]

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