Comparative Proteomics of Rat Olfactory Bulb Reveal Insights Into Susceptibility and Resilience to Chronic-Stress-Induced Depression or Anxiety

doi:10.21203/rs.3.rs-586884/v1

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Comparative Proteomics of Rat Olfactory Bulb Reveal Insights Into Susceptibility and Resilience to Chronic-Stress-Induced Depression or Anxiety

https://doi.org/10.21203/rs.3.rs-586884/v1

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As a main risk factor of both anxiety and depression, chronic stress can cause the abnormality of olfactory bulb (OB). However, the unique and common neurobiological underpinnings underlying the OB dysfunction of these two disorders are still poorly understood. Previously, we have built depression-susceptible (Dep-Sus), anxiety-susceptible (Anx-Sus) and insusceptible (Insus) groups through the use of a valid chronic mild stress (CMS) regime. To continuously explore the protein expression changes in these three groups, comparative quantitative proteomics analysis was conducted on the rat OB as crucial part of the olfactory system. Next, bioinformatics analyses were implemented whereas protein expressions were independently analyzed by parallel reaction monitoring (PRM) or Western blot (WB). The OB-proteome analysis identified totally 133 differentially expressed proteins as a CMS response. These deregulated proteins were involved in multiple functions and significant pathways potentially correlated with phenotypes of maladaptive behavior of depression or anxiety as well as adaptive behavior, and hence might act as potential candidate protein targets. The subsequent PRM-based or WB-based analyses showed that changes in Nefl, Mtmr7 and Tk2; Prkaca, Coa3, Cox6c2, Lamc1 and Tubal3; and Pabpn1, Nme3, Sos1 and Lum were uniquely associated with Dep-Sus, Anx-Sus, and Insus groups, respectively. This suggested that the identical CMS differently impacted the olfactory protein regulation system. To sum up, our present data as a useful proteomics underpinning provided the common and distinct molecular insights into the biochemical understanding of OB dysfunction underlying susceptibility and resiliency to chronic-stress-induced anxiety or depression.

Molecular Biology

Head & Neck Surgery

anxiety

chronic stress

depression

olfactory bulb

proteomics

In recent years, anxiety and depression are becoming two of the most predominant and devastating psychiatric diseases [1]. In general, they exhibit multiple and different clinical manifestations, however are high comorbid [2]. Currently, most data from clinical and basic studies are often mixed owing to high degree of overlap between anxiety and depression disorders in pathophysiology and comorbidity [3–5]. To some extent, this will result in the ambiguous knowledge of the regulatory factors of the two diseases [6]. To address the issue, recently some researchers set about separately investigating individuals with a single disorder in order to uncover the specific and common characteristics of anxiety and depression [7–10].

As two highly heterogeneous disorders, depression and anxiety shared the same risk factors, such as acute and chronic life event stress [11–13]. Chronic life stress is generally considered as detrimental environmental elements and can lead to the onset of anxiety and depression [14, 15]. In the preclinical research, there has been a widely used way, chronic mild stress (CMS), to mimic some socio-environmental factors affecting humans [13, 16]. When exposed to the CMS, some rodents develop the anxious and depressive behaviors, while others display a stress resilient feature [16]. For comprehensively understanding the potential neurobiological processes behind these CMS-induced behavioral profiles, the focus on the underlying neurochemical components will be very useful and meaningful.

It is known that olfaction is essential for a variety of behaviors, such as emotional regulation, food positioning, aggressive behavior and social and sexual behaviors [17, 18]. As the first central structure of the olfactory system in the brain, olfactory bulb (OB) projects to brain regions that closely related with cognition and emotion, such as hippocampus, amygdala and orbitofrontal cortex [19]. Multiple reports have evidenced that anxiety and depression lead to a decreased OB volume and olfactory sensitivity in animals and humans [20–23]. Despite the olfactory dysfunction correlates with both anxious and depressive symptoms [24], the underlying mechanisms may be different and remain unclear. In the case, identification of the unique and shared molecular patterns that underlie OB abnormality of adaptability and mal-adaptability to anxiety or depression has become necessary.

Previously, we applied the CMS paradigm to segregate and obtain depression-susceptible (Dep-Sus), anxiety-susceptible (Anx-Sus) and insusceptible (Insus) groups, and conducted the comparative proteomics analysis of the rat hippocampus [25]. In this work, we used the OB tissues from the same batch of our previous CMS rat model and continued to explore stress-related depressive and anxiety disorders [25]. The isobaric tags for relative and absolute quantitation (iTRAQ)-based comparative proteomics were carried out on the OB tissues of the control (Ctrl) and the three stressed groups. The present proteomic data could serve as a helpful molecular basis to better knowledge of the specificity and commonality of the molecular mechanisms that underlie chronic-stress-induced depression or anxiety and stress resilience.

CMS rat model

Adult male Sprague-Dawley rats (weighting about 250g) were provided by the Animal Facility of Chongqing Medical University. They were individually housed under standard conditions in the room temperature of 21-22°C, humidity 55 ± 5% with ad libitum feeding. The animals were maintained under a 12 h light/dark cycle. All experimental procedures were performed in accordance with the local Ethics Committee for the care and use of laboratory animals. As described in our previous study [25], the eight-week CMS procedure was used to develop the depressive and anxious rat model. The stressed rats were segregated into the three groups: (i) Dep-Sus group (evaluated by sucrose preference (SP) and forced swimming (FS) tests); (ii) Anx-Sus group (evaluated by elevated plus maze (EPM) test); and (iii) Insus group. Additional non-stressed rats acted as the Ctrl group. For more detailed information, please refer to our previous work [25].

Protein sample preparation for proteomics analysis

The animals were sacrificed after the behavioral assessments were finished. The OB tissues were rapidly collected on ice, then snap-frozen in liquid nitrogen and stored at −80°C until analysis. For protein extraction of OB tissues, SDT sample lysis buffer (4% SDS, 0.1 M dithiothreitol, 0.1 M Tris–HCl, pH 8.0) containing protease inhibitors was used. After the samples were boiled and centrifuged, Pierce bicinchoninic acid assay kit was used to determine the protein concentrations.

Protein digestion, peptide iTRAQ labeling and fractionation, and liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Protein digestion, peptide labeling and fractionation, as well as LC-MS/MS analysis were performed as previously described [26]. The classic filter-aided sample preparation-based method was employed for protein digestion with 10-kD ultrafiltration centrifuge tubes. The resulting peptides were labeled by using 8-plex iTRAQ reagents. As shown in Figure 1A, eight peptide samples from the Ctrl, Dep-Sus, Anx-Sus and Insus rats were labeled using the iTRAQ 8-plex kits 113-121. Each sample was mixed equally from two or three rats in each group [27]. After the iTRAQ labeling, the eight peptide samples were combined. The high-pH reversed-phase liquid chromatography (RPLC) was used to off-line separate the peptide mixture. The elution of the RPLC fractionation was performed according to our previously described procedure [26].

The obtained fractions were desalted, dried and re-constituted in 0.1% formic acid. After that, the solutions were then loaded into a C18 nanoViper trapping column (3 μm, 100 Å). The peptides were online separated using an Easy-nLC 1200 system (ThermoFisher) equipped with a C18 analytical column (50 μm × 150 mm, 3 μm, 100 Å). We used a 50 min elution gradient by linearly increasing the solution B (80% acetonitrile/0.1% formic acid) from 8 to 38%. For the MS analysis, a Q-Exactive Orbitrap mass spectrometer (ThermoFisher) coupled with a Nano Flex ion source was utilized as previously described [26]. A data-dependent acquisition mode was used to acquire the tandem MS data. A higher-energy collision dissociation was conducted to analyze the MS spectra with two and above charge-state.

Peptide identification and quantification

As depicted previously [26], the MS/MS spectra were extracted and searched using the Proteome Discoverer (version 2.1) and the Sequest HT search engine (ThermoFisher) accepting common fixed and variable modifications and two trypsin missed cleavages. The UniProt database was meanwhile imported into the software. Precursor fragment mass tolerance was ±10 ppm and fragment mass tolerance was ±0.05 Da. Peptide and protein identifications were performed through the corresponding decoy database and a false discovery rate (FDR) below 1%. The relative ratios of peptides and proteins across samples was computed using Reporter Ions Quantifier and Peptide and Protein Quantifier nodes (ThermoFisher). The iTRAQ-based protein ratios were further analyzed by using a two-tailed Student’s t-test. The differential proteins showing 1.2-fold changes along with a p-value lower than 0.05 were considered as significant. The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD023766 [28].

Bioinformatics

The differential protein data were analyzed using the OmicsBean tool (http://www.omicsbean.cn/) to obtain and enrich the Gene Ontology (GO) terms, including biological processes (GO-BP), molecular functions (GO-MF), and cellular components (GO-CC). Meanwhile, we used a Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg/) to identify the significant pathways with p-values of lower than 0.1 as depicted previously [29]. Further, protein-protein interaction (PPI) was analyzed by using the database of the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING).

Parallel reaction monitoring (PRM) analysis

For the PRM analysis, the protein and peptide sample preparations were conducted following the iTRAQ-based proteomic experiment. The tryptic peptides were analyzed through the use of the Q-Exactive mass spectrometer. The peptides were fragmented using a twenty-eight normalized collision energy, and the resultant fragments were further detected in the Orbitrap at a 35,000 resolution. The acquired MS raw data were analyzed by use of the Proteome Discoverer and processed with the software Skyline (version 19.1).

Western blot (WB) analysis

The WB was conducted on protein extracts of rat OB tissues that were also used for the iTRAQ experiment. Our previous study served as a prototype when carried out the procedures of electrophoresis, transfer and immunodetection [13, 30]. The primary antibodies used in our experiments were as follows: Lamc1 (1:2,000; 67706-1-Ig, Proteintech), Mtor (1:12,000; 66888-1-Ig, Proteintech), Homer3 (1:800; 16624-1-AP, Proteintech), Lum (1:800; D122637, Sangon), Tubal3 (1:1,000; D199287, Sangon), GAPDH (1:5,000; 60004-1-Ig, Proteintech) and β-tubulin (1:6,000; 10094-1-AP, Proteintech). Anti-mouse (1:10,000; D110087, Sangon) or anti-rabbit (1:10,000; D110058, Sangon) horseradish peroxidase-conjugated IgG was used as secondary antibody. After immunodetection, the intensities of the immunostained bands were normalized by the intensity of the GAPDH or β-tubulin band. The blot images were analyzed using Quantity One analysis software (Bio-Rad).

Statistical analysis

All data were analyzed using Student’s t-test and the level of statistical significance for each analysis was set at p-values lower than 0.05. All data were expressed as the means ± standard error.

Comparative quantitative analysis of OB proteomes of the CMS model rats

In this work, the OB samples used were from the same batch of stress-induced model rats in our previous work [25]. To sum up, SP and FS tests were firstly applied to assess the stress-induced depressive-like behavior such as anhedonia and behavioral despair. Subsequently, the EPM test was used to indicate the anxious-like behavior. On the basis of these tests, we obtained a subset of the Dep-Sus, Anx-Sus, Insus, and Ctrl groups. To some extent, our developed preclinical model was effective and useful for investigating the neurobiological components related with the susceptibility and adaptability of stress-induced anxiety or depression.

To determine the OB site-specific proteomic signature, an iTRAQ-based shotgun quantitation approach was applied to assess protein expression changes in the OB tissues derived from the Ctrl, Dep-Sus, Anx-Sus and Insus groups (Figure 1A). Five rats per group were used in this proteomics experiment, and the OB protein extracts from two or three rats were equally mixed for each sample [27]. The comparative proteomics identified and quantified 3,526 non-redundant proteins based on FDR lower than 0.01 (Supplemental Table S1). Comparing the OB proteomes of the Ctrl and the three stressed groups, a total of 133 stress-responsive proteins tended to be significantly abnormally expressed.

CMS-responsive deregulated protein function and interaction network features

By comparative analysis of OB proteomes, in the Dep-Sus group 12 proteins were found to be downregulated and 12 proteins were overexpressed. In the Anx-Sus group 18 down-regulated and 26 overexpressed, and in the Insus group 23 down-regulated and 64 overexpressed. Eight proteins along with similar disturbances were seen in the two CMS-susceptible groups, potentially representing the shared components of anxious-like and depressive-like behavioral phenotypes. Among the insusceptible and the two susceptible groups, we also observed that 14 proteins were similar de-regulations and might be as a result of stress (Figure 2A). Additionally, the deregulated proteins distinctly associated with the three phenotypes accounted for 72%. This indicated that the three stressed groups had different protein change profiles as a stress response. The phenotype-specific functional profiles of the OB proteins were evidenced by the use of unsupervised hierarchical clustering of 133 deregulated proteins, as shown in Figure 2B.

Next, these deregulated proteins in the three groups were analyzed through the use of the OmicsBean software to have a better knowledge of phenotype-associated neurobiological functions and pathways. In the Dep-Sus group, enrichment analysis of GO and KEGG pathway was carried out on the 24 deregulated proteins (Supplemental Table S2). The GO-BP, GO-CC and GO-MF categories were recognized to be significantly enriched, which were 350, 64 and 61 terms respectively. Here the top 10 enriched GO terms were displayed (Figure 2C). The GO-BP enrichment showed that most proteins were involved in positive regulation of biological process, transport, endocytosis, response to external stimulus, and adaptive immune response. Many proteins in the GO-CC category belonged to peroxisomal matrix, microbody lumen, plasma membrane and cytosol. GO-MF category analysis indicated that most proteins were involved in receptor, antigen and beta-endorphin binding, transporter and enzyme activity. KEGG-based enrichment indicated that these deregulated proteins were primarily involved in 41 significant pathways such as African trypanosomiasis, malaria, endocytosis and multiple regulating and signaling pathways (Figure 3A).

Meanwhile, the 44 proteins deregulated in the Anx-Sus group were subjected to enrichment analysis of GO and KEGG pathway. The GO-BP, GO-CC and GO-MF categories were recognized to be remarkably overrepresented, which were 638, 164 and 115 terms. The top ten overrepresented GO terms were showed (Figure 2D). The GO-BP classification analysis indicated that many proteins were associated with fibril, extracellular matrix and structure, intermediate filament and protein complex subunit organizations. GO-CC category analysis displayed that most proteins belonged to polymeric cytoskeletal and supramolecular fibers, organelle, cytoplasmic and cytoskeletal parts, and cytosol. GO-MF indicated most of the deregulated proteins were correlated with protein and complex binding, structural molecule and protein heterodimerization and dimerization activities. The KEGG enrichment analysis revealed these deregulated proteins were primarily involved in 70 significant pathways including metabolism, signaling, regulating, apoptosis and synapse pathways (Figure 3B).

Afterwards, GO and KEGG pathway terms of 87 deregulated proteins from the Insus group were also enriched. We identified 771 GO-BP, 187 GO-CC and 110 GO-MF terms to be remarkably overrepresented. The top 10 enriched GO items were displayed in Figure 2E. GO-BP enrichment exhibited that most of the deregulated proteins were involved in organonitrogen compound and cellular protein metabolic processes, organism and cellular processes, and nervous system and cell development. GO-CC analysis showed that many proteins belonged to cytoplasmic, organelle and intracellular parts, and cytosol. GO-MF category analysis showed most proteins were predicted to be involved in protein, poly(A) RNA and glutathione binding, and KEGG-based analysis showed that the deregulated proteins were primarily associated with 43 significant pathways such as choline, inositol phosphate and glycerophospholipid metabolisms, multiple signaling/regulating/synapse pathways (Figure 3C).

On the whole, of these significantly-enriched KEGG pathways, we found that there were 21 shared items between the three groups (Figure 3D). Between the two susceptible groups, the 26 pathways were common. Importantly, the 11, 32 and 6 pathways were found to be specifically correlated with the Dep-Sus, Anx-Sus and Insus groups, respectively, potentially suggesting the three different neurobiological response to stress.

To further examine the comprehensive information from the proteomic data of the three groups, the protein-protein interaction (PPI) networks were mapped and then analyzed. The deregulated proteins connected with the significantly enriched KEGG pathways were merged to construct the networks as shown in Figure 4A-C. On the basis of a unified conceptual framework, a total of 10, 33, and 50 proteins were identified to be significant nodes in the mapped networks from the three stressed groups (Dep-Sus, Anx-Sus and Insus, respectively). These generated PPI networks indicated that there was a direct linkage between the deregulated proteins and significantly overrepresented KEGG pathways, and therefore offered a phenotype-associated interactome pool.

PRM or WB analyses of CMS-responsive proteins

In this work, we further independently investigated 16 deregulated proteins of interest from the significant pathways and networks using the PRM-based or WB-based quantitative methods. It could be seen that the PRM and WB data basically mirrored the results from the iTRAQ experiment (Supplemental Figure S1). There are some objective discrepancies between these quantitative data, as depicted in other proteomic work [31-34]. Besides the detection difference of these methods [33, 34], the additional sample mixing procedure in the iTRAQ experiment was another possible cause. As compared to the Ctrl group, the expressions of Nefl, Mtmr7 and Tk2 were significantly down-regulated in the Dep-Sus group; the expressions of Prkaca, Coa3, Cox6c2, Lamc1 and Tubal3 were significantly up-regulated in the Anx-Sus group; and the expressions of Pabpn1 and Lum were significantly down-regulated while Nme3 and Sos1 were up-regulated in the Insus group (Figure 4D). Additionally, a reduced level of Homer3 in both the Dep-Sus and Anx-Sus groups, an elevated level of Pip4p2 in both the Anx-Sus and Insus groups, and a reduced level of Gh1 in the three groups were observed compared with the Ctrl group. The expression level of Mtor were also observed to be significantly down-regulated whereas up-regulated in both the Anx-Sus and Insus groups when compared to the Ctrl group.

In recent years, researchers have been focused on exploring the neurobiology of stress vulnerability and adaptability in anxiety and depression studies, however, these mechanisms are still unclear. To characterize the neurobiological pathways involved in anxious-like and depressive-like behaviors or resilience of rats at the protein expression level, CMS as a classic model was employed to generate the three stress-response phenotypes (Dep-Sus, Anx-Sus and Insus) in our previous study [25]. This provided a useful preclinical model for study the specificity and commonality of depression and anxiety, thus contributing to the translational biomedical research.

Several previous studies observed that CMS resulted in a decreased OB volume and olfactory dysfunction in the animal model [22, 23, 35]. To investigate a global protein expression within the OB tissues of the Dep-Sus, Anx-Sus and Insus rats, the iTRAQ relative proteomics approach was utilized. Comparing the Ctrl and three stressed groups, a total of 133 CMS-response proteins were identified in the OB tissue. The overlapped proteins between the two susceptible subgroups suggested a potential commonality of depression and anxiety. The similar deregulations between the two susceptible subgroups and the insusceptible subgroup might only reflect a CMS-response result. Interestingly, the deregulated proteins uniquely in the three stressed cohorts might presented CMS-induced behavioral phenotype differences. The different protein dysfunctional profiles of the three stressed subgroups was further exhibited and evidenced through the use of the clustering analysis. The proteomic data would help us identify potential biomarkers of behavioral phenotypes resembling depressive and anxious behaviors and the resilience to stress.

Next, these deregulated proteins were subjected to bioinformatics investigation, suggesting many affected biological processes and pathways in the rat OB region distinctly correlated with the three stress-induced behavioral phenotypes. This reflected potential differences in important deregulation systems and active biological pathways that happened in these chronically stressed groups, demonstrating that the rats exhibited varied neurobiological responses to the same stressful stimuli. Afterwards, the PPI networks captured the protein deregulation systems coupled with some important KEGG pathways, potentially offering some valuable information for the phenotype-correlated molecular underpinnings in the OB site.

Further, there were several important deregulated proteins that involving with the significant pathways. In this study, we used PRM-based or WB-based quantitative methods to further independently analyze them. The results showed that Nefl, Mtmr7 and Tk2 were specifically deregulated in the OB of the Dep-Sus group, while Prkaca, Coa3, Cox6c2, Lamc1 and Tubal3 were specifically deregulated in the Anx-Sus group. The specificity of these protein expressions in the OB suggested that the identical CMS resulted in the different molecular responses, thereby offering an impetus to diverse neurobiological processes along with maladaptive behavior of depression or anxiety. Interestingly, the expressions of Pabpn1, Lum, Nme3 and Sos1 were found to be specifically deregulated in the Insus group. These specific changes might be several potential molecular adaptions in the OB to positively cope with the stress-caused dysfunctions for rat adaptive behavior [11, 13].

We found that these phenotype-specific deregulated proteins were primarily involved in multiple metabolic, signaling and synapse pathways. Cox6c2 is engaged in oxidative phosphorylation, and Mtmr7, Tk2 and Nme3 are involved in some metabolic pathways such as inositol phosphate and pyrimidine metabolisms. The altered metabolic processes along with abnormal oxidative phosphorylation system in the OB would lead to an energy imbalance of the olfactory system [35, 36]. Lamc1 and Sos1 are involved in PI3K-Akt signaling, a pathway that has been reported to be strongly associated with stress-related disorders [37, 38]. In general, chronic stress causes impairment of synaptic morphological and functional plasticity, which can be restored by antidepressants such as ketamine via multiple intracellular pathways including PI3K-Akt [37, 39, 40]. These altered proteins may be potential mediators of PI3K-Akt signaling for regulating stress behaviors of the rats, and thus helpful for drug development for the disease treatment [41]. In addition, Prkaca is the catalytic subunit alpha of protein kinase A that regulates intracellular signal transduction cascades connected to G-coupled receptors [42]. The dysregulation of Prkaca in the Anx-Sus group potentially played a key role on the anxiety-like behavior via the kinase signaling [43]. Interestingly, Homer3 is a scaffold protein involved in the binding of signaling molecules at the postsynaptic densities and related to depression [44]. The reduced level of Homer3 was previously found in the hippocampus of a genetic rat model showing depressive-like behavior and reversed by glutamatergic antidepressant ketamine [45]. Here we found that the expression of Homer3 was significantly down-regulated in the OB of the two CMS-susceptible groups. The result suggested that Homer3 might be a potential therapeutic target for both anxiety and depression therapy. Taken together, the protein expressions specifically connected with the three phenotypes could offer a more nuanced underpinning for future research. It is also important to further investigate the detailed mechanisms behind these protein aberrations in the OB of the stressed groups.

In conclusion, a shotgun quantitation approach based on iTRAQ and PRM was used to evaluate the expression alterations of the rat OB proteins caused by CMS. Our protein abundance change profiles identified some OB protein candidates that could serve as potential biomarkers of phenotypes resembling maladaptive behavior of depression or anxiety as well as adaptive behavior. The findings might be helpful for revealing the underlying mechanisms of stress-related disorders. The present data as a valuable proteomics underpinning could enhance our knowledge of the similarities and differences of chronic-stress-induced anxiety/depression and stress resistance concerning olfactory dysfunction.

CMS: Chronic mild stress; EPM: Elevated plus maze; FS: Forced swimming; GO, Gene ontology; iTRAQ: Isobaric tags for relative and absolute quantitation; LC-MS/MS: Liquid chromatography-tandem mass spectrometry; OB: Olfactory bulb; PRM: Parallel reaction monitoring; SP: Sucrose preference.

Acknowledgments

Not applicable.

Authors’ contributions

CF, JC and JZ designed the project. DL, XC and LW performed experiments. DL, LW, WL, FY and RH analyzed the data. CF, JC and JZ prepared the manuscript. All authors read and approved the final manuscript.

Funding

This study was financially supported by the National Natural Science Foundation of China (grant numbers 31770890, 31860276 and 31570826).

Availability of data and materials

The proteomics raw data can be found in the ProteomeXchange Consortium with the identifier PXD023766. The datasets supporting this article have been uploaded as part of the electronic supplementary materials.

Ethics approval and consent to participate

The experimental protocol was approved by the Ethical Committee of Chongqing Medical University. Animals were treated in accordance with the National Institutes of Health Guidelines for the use and care of laboratory animals.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Comparative Proteomics of Rat Olfactory Bulb Reveal Insights Into Susceptibility and Resilience to Chronic-Stress-Induced Depression or Anxiety

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