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Hahn, M. & Ahlgren, U. (2023). 3D optical molecular imaging of the rodent pancreas by OPT and LSFM. In: Anna Moore; Ping Wang (Ed.), Type-1 diabetes: methods and protocols (pp. 1-19). New York: Humana Press
Open this publication in new window or tab >>3D optical molecular imaging of the rodent pancreas by OPT and LSFM
2023 (English)In: Type-1 diabetes: methods and protocols / [ed] Anna Moore; Ping Wang, New York: Humana Press, 2023, , p. 19p. 1-19Chapter in book (Refereed)
Abstract [en]

The rodent pancreas is the prevalent model system for preclinical diabetes research. However, due to the compound endocrine–exocrine organization of the gland, with the endocrine islets of Langerhans scattered by the thousands throughout the much greater exocrine parenchyma, stereological assessments of endocrine cell mass, commonly insulin-producing ß-cells, are exceedingly challenging. In recent years, optical mesoscopic imaging techniques such as optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) have seen dramatic developments, enabling 3D visualization of fluorescently labeled cells in mm- to cm-sized tissues with μm resolution. Here we present a protocol for 3D visualization and “absolute” quantitative assessments of, for example, islet mass throughout the volume of rodent pancreata with maintained spatial context.

Place, publisher, year, edition, pages
New York: Humana Press, 2023. p. 19
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2592
Keywords
Diabetes, Insulin, Islets of Langerhans, Light sheet fluorescence microscopy, Optical clearing, Optical projection tomography, Pancreas, Rodents, Whole mount immunohistochemistry
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-202060 (URN)10.1007/978-1-0716-2807-2_1 (DOI)36507982 (PubMedID)2-s2.0-85144475952 (Scopus ID)978-1-0716-2806-5 (ISBN)978-1-0716-2809-6 (ISBN)978-1-0716-2807-2 (ISBN)
Available from: 2023-01-03 Created: 2023-01-03 Last updated: 2023-03-23Bibliographically approved
Chotiwan, N., Rosendal, E., Willekens, S. M. A., Schexnaydre, E., Nilsson, E., Lindquist, R., . . . Överby, A. K. (2023). Type I interferon shapes brain distribution and tropism of tick-borne flavivirus. Nature Communications, 14(1), Article ID 2007.
Open this publication in new window or tab >>Type I interferon shapes brain distribution and tropism of tick-borne flavivirus
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2023 (English)In: Nature Communications, E-ISSN 2041-1723, Vol. 14, no 1, article id 2007Article in journal (Refereed) Published
Abstract [en]

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar-/- mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar-/-, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

Place, publisher, year, edition, pages
Springer Nature, 2023
National Category
Microbiology in the medical area Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Neurosciences
Identifiers
urn:nbn:se:umu:diva-206780 (URN)10.1038/s41467-023-37698-0 (DOI)000967732600009 ()37037810 (PubMedID)2-s2.0-85152115180 (Scopus ID)
Funder
The Kempe Foundations, SMK-1532Knut and Alice Wallenberg Foundation, KAW2015.0284Swedish Research Council, 2018-05851Swedish Research Council, 2017-01307Swedish Research Council, 2020-06224Swedish Research Council, 2021-06602
Available from: 2023-04-24 Created: 2023-04-24 Last updated: 2023-09-05Bibliographically approved
Hahn, M., Nord, C., van Krieken, P. P., Berggren, P.-O., Ilegems, E., Cheddad, A. & Ahlgren, U. (2022). Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes. Scientific Data, 9, Article ID 558.
Open this publication in new window or tab >>Quantitative 3D OPT and LSFM datasets of pancreata from mice with streptozotocin-induced diabetes
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2022 (English)In: Scientific Data, E-ISSN 2052-4463, Vol. 9, article id 558Article in journal (Refereed) Published
Abstract [en]

Mouse models for streptozotocin (STZ) induced diabetes probably represent the most widely used systems for preclinical diabetes research, owing to the compound’s toxic effect on pancreatic β-cells. However, a comprehensive view of pancreatic β-cell mass distribution subject to STZ administration is lacking. Previous assessments have largely relied on the extrapolation of stereological sections, which provide limited 3D-spatial and quantitative information. This data descriptor presents multiple ex vivo tomographic optical image datasets of the full β-cell mass distribution in mice subject to single high and multiple low doses of STZ administration, and in glycaemia recovered mice. The data further include information about structural features, such as individual islet β-cell volumes, spatial coordinates, and shape as well as signal intensities for both insulin and GLUT2. Together, they provide the most comprehensive anatomical record of the effects of STZ administration on the islet of Langerhans in mice. As such, this data descriptor may serve as reference material to facilitate the planning, use and (re)interpretation of this widely used disease model.

Place, publisher, year, edition, pages
Nature Publishing Group, 2022
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-193538 (URN)10.1038/s41597-022-01546-5 (DOI)000852384000002 ()36088402 (PubMedID)2-s2.0-85138129001 (Scopus ID)
Funder
Swedish Diabetes AssociationUmeå UniversityFamiljen Erling-Perssons StiftelseEU, FP7, Seventh Framework Programme, 289932EU, FP7, Seventh Framework Programme, 613879Knut and Alice Wallenberg FoundationSwedish Research CouncilNovo Nordisk
Note

Originally included in thesis in manuscript form.

Available from: 2022-04-06 Created: 2022-04-06 Last updated: 2022-10-03Bibliographically approved
Hahn, M., Nord, C., Eriksson, M., Morini, F., Alanentalo, T., Korsgren, O. & Ahlgren, U. (2021). 3D imaging of human organs with micrometer resolution - applied to the endocrine pancreas. Communications Biology, 4(1), Article ID 1063.
Open this publication in new window or tab >>3D imaging of human organs with micrometer resolution - applied to the endocrine pancreas
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2021 (English)In: Communications Biology, E-ISSN 2399-3642, Vol. 4, no 1, article id 1063Article in journal (Refereed) Published
Abstract [en]

The possibility to quantitatively study specific molecular/cellular features of complete human organs with preserved spatial 3D context would have widespread implications for pre-clinical and clinical medicine. Whereas optical 3D imaging approaches have experienced a formidable revolution, they have remained limited due to current incapacities in obtaining specific labelling within large tissue volumes. We present a simple approach enabling reconstruction of antibody labeled cells within entire human organs with preserved organ context. We demonstrate the utility of the approach by providing volumetric data and 3D distribution of hundreds of thousands of islets of Langerhans within the human pancreas. By assessments of pancreata from non-diabetic and type 2 diabetic individuals, we display previously unrecognized features of the human islet mass distribution and pathology. As such, this method may contribute not only in unraveling new information of the pancreatic anatomy/pathophysiology, but it may be translated to essentially any antibody marker or organ system.

Place, publisher, year, edition, pages
Springer Nature, 2021
National Category
Cell and Molecular Biology Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-187770 (URN)10.1038/s42003-021-02589-x (DOI)000694906000003 ()2-s2.0-85114856810 (Scopus ID)
Available from: 2021-09-22 Created: 2021-09-22 Last updated: 2023-03-24Bibliographically approved
Davies, W. I. L., Sghari, S., Upton, B. A., Nord, C., Hahn, M., Ahlgren, U., . . . Gunhaga, L. (2021). Distinct opsin 3 (Opn3) expression in the developing nervous system during mammalian embryogenesis. eNeuro, 8(5), Article ID ENEURO.0141-21.2021.
Open this publication in new window or tab >>Distinct opsin 3 (Opn3) expression in the developing nervous system during mammalian embryogenesis
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2021 (English)In: eNeuro, E-ISSN 2373-2822, Vol. 8, no 5, article id ENEURO.0141-21.2021Article in journal (Refereed) Published
Abstract [en]

Opsin 3 (Opn3) is highly expressed in the adult brain, however, information for spatial and temporal expression patterns during embryogenesis is significantly lacking. Here, an Opn3-eGFP reporter mouse line was used to monitor cell body expression and axonal projections during embryonic and early postnatal to adult stages. By applying 2D and 3D fluorescence imaging techniques, we have identified the onset of Opn3 expression, which predominantly occurred during embryonic stages, in various structures during brain/head development. In ad-dition, this study defines over twenty Opn3-eGFP-positive neural structures never reported before. Opn3-eGFP was first observed at E9.5 in neural regions, including the ganglia that will ultimately form the trigeminal, facial and vestibulocochlear cranial nerves (CNs). As development proceeds, expanded Opn3-eGFP expression coincided with the formation and maturation of critical components of the central and peripheral nervous systems (CNS, PNS), including various motor-sensory tracts, such as the dorsal column-medial lemniscus (DCML) sensory tract, and olfactory, acoustic, and optic tracts. The widespread, yet distinct, detection of Opn3-eGFP already at early embryonic stages suggests that Opn3 might play important functional roles in the developing brain and spinal cord to regulate multiple motor and sensory circuitry systems, including proprio-ception, nociception, ocular movement, and olfaction, as well as memory, mood, and emotion. This study presents a crucial blueprint from which to investigate autonomic and cognitive opsin-dependent neural development and resultant behaviors under physiological and pathophysiological conditions.

Place, publisher, year, edition, pages
Society for Neuroscience, 2021
Keywords
Brain, Development, Encephalopsin, Nervous system, Opn3, OPT
National Category
Neurosciences Developmental Biology
Identifiers
urn:nbn:se:umu:diva-187852 (URN)10.1523/ENEURO.0141-21.2021 (DOI)000704430100013 ()34417283 (PubMedID)2-s2.0-85114912713 (Scopus ID)
Funder
Swedish Research Council, 2017-01430The Kempe Foundations, SMK-1763Swedish Research Council, 2017-01307
Available from: 2021-09-23 Created: 2021-09-23 Last updated: 2023-09-05Bibliographically approved
Serra-Navarro, B., Fernandez-Ruiz, R., García-Alamán, A., Pradas-Juni, M., Fernandez-Rebollo, E., Esteban, Y., . . . Gasa, R. (2021). Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass. Molecular Metabolism, 53, Article ID 101264.
Open this publication in new window or tab >>Gsα-dependent signaling is required for postnatal establishment of a functional β-cell mass
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2021 (English)In: Molecular Metabolism, ISSN 2212-8778, Vol. 53, article id 101264Article in journal (Refereed) Published
Abstract [en]

Objective: Early postnatal life is a critical period for the establishment of the functional β-cell mass that will sustain whole-body glucose homeostasis during the lifetime. β cells are formed from progenitors during embryonic development but undergo significant expansion in quantity and attain functional maturity after birth. The signals and pathways involved in these processes are not fully elucidated. Cyclic adenosine monophosphate (cAMP) is an intracellular signaling molecule that is known to regulate insulin secretion, gene expression, proliferation, and survival of adult β cells. The heterotrimeric G protein Gs stimulates the cAMP-dependent pathway by activating adenylyl cyclase. In this study, we sought to explore the role of Gs-dependent signaling in postnatal β-cell development.

Methods: To study Gs-dependent signaling, we generated conditional knockout mice in which the α subunit of the Gs protein (Gsα) was ablated from β-cells using the Cre deleter line Ins1Cre. Mice were characterized in terms of glucose homeostasis, including in vivo glucose tolerance, glucose-induced insulin secretion, and insulin sensitivity. β-cell mass was studied using histomorphometric analysis and optical projection tomography. β-cell proliferation was studied by ki67 and phospho-histone H3 immunostatining, and apoptosis was assessed by TUNEL assay. Gene expression was determined in isolated islets and sorted β cells by qPCR. Intracellular cAMP was studied in isolated islets using HTRF-based technology. The activation status of the cAMP and insulin-signaling pathways was determined by immunoblot analysis of the relevant components of these pathways in isolated islets. In vitro proliferation of dissociated islet cells was assessed by BrdU incorporation.

Results: Elimination of Gsα in β cells led to reduced β-cell mass, deficient insulin secretion, and severe glucose intolerance. These defects were evident by weaning and were associated with decreased proliferation and inadequate expression of key β-cell identity and maturation genes in postnatal β-cells. Additionally, loss of Gsα caused a broad multilevel disruption of the insulin transduction pathway that resulted in the specific abrogation of the islet proliferative response to insulin.

Conclusion: We conclude that Gsα is required for β-cell growth and maturation in the early postnatal stage and propose that this is partly mediated via its crosstalk with insulin signaling. Our findings disclose a tight connection between these two pathways in postnatal β cells, which may have implications for using cAMP-raising agents to promote β-cell regeneration and maturation in diabetes.

Place, publisher, year, edition, pages
Elsevier, 2021
Keywords
cAMP, Cell maturation, Gs, Insulin signaling, Postnatal development, Replication, β-Cell mass
National Category
Endocrinology and Diabetes Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-191152 (URN)10.1016/j.molmet.2021.101264 (DOI)000704022000004 ()34091063 (PubMedID)2-s2.0-85108283081 (Scopus ID)
Available from: 2022-01-10 Created: 2022-01-10 Last updated: 2022-01-10Bibliographically approved
Quilichini, E., Fabre, M., Nord, C., Dirami, T., Le Marec, A., Cereghini, S., . . . Haumaitre, C. (2021). Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease. Journal of Pathology, 254(1), 31-45
Open this publication in new window or tab >>Insights into the etiology and physiopathology of MODY5/HNF1B pancreatic phenotype with a mouse model of the human disease
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2021 (English)In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 254, no 1, p. 31-45Article in journal (Refereed) Published
Abstract [en]

Maturity‐onset diabetes of the young type 5 (MODY5) is due to heterozygous mutations or deletion of HNF1B. No mouse models are currently available to recapitulate the human MODY5 disease. Here, we investigate the pancreatic phenotype of a unique MODY5 mouse model generated by heterozygous insertion of a human HNF1B splicing mutation at the intron‐2 splice donor site in the mouse genome. This Hnf1bsp2/+ model generated with targeted mutation of Hnf1b mimicking the c.544+1G>T (<IVS2nt+1G>T) mutation identified in humans, results in alternative transcripts and a 38% decrease of native Hnf1b transcript levels. As a clinical feature of MODY5 patients, the hypomorphic mouse model Hnf1bsp2/+ displays glucose intolerance. Whereas Hnf1bsp2/+ isolated islets showed no altered insulin secretion, we found a 65% decrease in pancreatic insulin content associated with a 30% decrease in total large islet volume and a 20% decrease in total β‐cell volume. These defects were associated with a 30% decrease in expression of the pro‐endocrine gene Neurog3 that we previously identified as a direct target of Hnf1b, showing a developmental etiology. As another clinical feature of MODY5 patients, the Hnf1bsp2/+ pancreases display exocrine dysfunction with hypoplasia. We observed chronic pancreatitis with loss of acinar cells, acinar‐to‐ductal metaplasia, and lipomatosis, with upregulation of signaling pathways and impaired acinar cell regeneration. This was associated with ductal cell deficiency characterized by shortened primary cilia. Importantly, the Hnf1bsp2/+ mouse model reproduces the pancreatic features of the human MODY5/HNF1B disease, providing a unique in vivo tool for molecular studies of the endocrine and exocrine defects and to advance basic and translational research.

Place, publisher, year, edition, pages
John Wiley & Sons, 2021
Keywords
exocrine dysfunction, glucose intolerance, haploinsufficiency, HNF1B, maturity-onset diabetes of the young (MODY), optical projection tomography (OPT), pancreatic hypoplasia, pancreatitis, primary cilia, β-cells
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-181833 (URN)10.1002/path.5629 (DOI)000630230000001 ()33527355 (PubMedID)2-s2.0-85102621816 (Scopus ID)
Available from: 2021-04-06 Created: 2021-04-06 Last updated: 2022-04-19Bibliographically approved
Eriksson, M., Litwak, S. A., Yun, Y., Stanley, W. J., Thorn, P., Ahlgren, U. & Gurzov, E. N. (2021). Insulin-Binding Peptide Probes Provide a Novel Strategy for Pancreatic β-Cell Imaging. Molecular Pharmaceutics, 18(12), 4428-4436
Open this publication in new window or tab >>Insulin-Binding Peptide Probes Provide a Novel Strategy for Pancreatic β-Cell Imaging
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2021 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 18, no 12, p. 4428-4436Article in journal (Refereed) Published
Abstract [en]

Type 1 diabetes develops in childhood and adolescence, with peak incidence in the early teenage years. There is an urgent need for an accurate method to detect insulin-producing β-cells in patients that is not affected by alterations in β-cell function. As part of our research program to design specific probes to measure β-cell mass, we recently developed a novel insulin-binding peptide probe (IBPP) for the detection of β-cells in vivo. Here, we applied our innovative method to show specific labeling of this IBPP to human and mouse fixed β-cells in pancreatic islets. Importantly, we showed staining of human and mouse islets in culture without any negative functional or cell viability impact. Moreover, the IBPP-stained mouse islets after tail vein injection in vivo, albeit with batch differences in staining efficiency. In conclusion, we provide evidence showing that the IBPP can be used for future accurate detection of β-cell mass in a variety of preclinical models of diabetes.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2021
Keywords
glucose-stimulated insulin secretion, insulin-binding peptide, pancreatic islets, type 1 diabetes, β-cell imaging
National Category
Endocrinology and Diabetes Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-189219 (URN)10.1021/acs.molpharmaceut.1c00616 (DOI)000755047200018 ()2-s2.0-85118183928 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 289932Swedish Research CouncilNovo Nordisk
Available from: 2021-11-10 Created: 2021-11-10 Last updated: 2023-09-05Bibliographically approved
Alanentalo, T., Hahn, M., Willekens, S. M. A. & Ahlgren, U. (2021). Mesoscopic Optical Imaging of the Pancreas: Revisiting Pancreatic Anatomy and Pathophysiology. Frontiers in Endocrinology, 12, Article ID 633063.
Open this publication in new window or tab >>Mesoscopic Optical Imaging of the Pancreas: Revisiting Pancreatic Anatomy and Pathophysiology
2021 (English)In: Frontiers in Endocrinology, E-ISSN 1664-2392, Vol. 12, article id 633063Article, review/survey (Refereed) Published
Abstract [en]

The exocrine-endocrine multipart organization of the pancreas makes it an exceedingly challenging organ to analyze, quantitatively and spatially. Both in rodents and humans, estimates of the pancreatic cellular composition, including beta-cell mass, has been largely relying on the extrapolation of 2D stereological data originating from limited sample volumes. Alternatively, they have been obtained by low resolution non-invasive imaging techniques providing little detail regarding the anatomical organization of the pancreas and its cellular and/or molecular make up. In this mini-review, the state of the art and the future potential of currently existing and emerging high-resolution optical imaging techniques working in the mm-cm range with μm resolution, here referred to as mesoscopic imaging approaches, will be discussed regarding their contribution toward a better understanding of pancreatic anatomy both in normal conditions and in the diabetic setting. In particular, optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) imaging of the pancreas and their associated tissue processing and computational analysis protocols will be discussed in the light of their current capabilities and future potential to obtain more detailed 3D-spatial, quantitative, and molecular information of the pancreas.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2021
Keywords
diabetes, light sheet fluorescence microscopy, mesoscopic imaging, optical projection tomography, pancreas
National Category
Endocrinology and Diabetes
Identifiers
urn:nbn:se:umu:diva-182014 (URN)10.3389/fendo.2021.633063 (DOI)000629989000001 ()2-s2.0-85102893401 (Scopus ID)
Available from: 2021-04-06 Created: 2021-04-06 Last updated: 2024-01-17Bibliographically approved
Hahn, M., Nord, C., Franklin, O., Alanentalo, T., Isaksson Mettävainio, M., Morini, F., . . . Ahlgren, U. (2020). Mesoscopic 3D imaging of pancreatic cancer and Langerhans islets based on tissue autofluorescence. Scientific Reports, 10(1), Article ID 18246.
Open this publication in new window or tab >>Mesoscopic 3D imaging of pancreatic cancer and Langerhans islets based on tissue autofluorescence
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2020 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 18246Article in journal (Refereed) Published
Abstract [en]

The possibility to assess pancreatic anatomy with microscopic resolution in three dimensions (3D) would significantly add to pathological analyses of disease processes. Pancreatic ductal adenocarcinoma (PDAC) has a bleak prognosis with over 90% of the patients dying within 5 years after diagnosis. Cure can be achieved by surgical resection, but the efficiency remains drearily low. Here we demonstrate a method that without prior immunohistochemical labelling provides insight into the 3D microenvironment and spread of PDAC and premalignant cysts in intact surgical biopsies. The method is based solely on the autofluorescent properties of the investigated tissues using optical projection tomography and/or light-sheet fluorescence microscopy. It does not interfere with subsequent histopathological analysis and may facilitate identification of tumor-free resection margins within hours. We further demonstrate how the developed approach can be used to assess individual volumes and numbers of the islets of Langerhans in unprecedently large biopsies of human pancreatic tissue, thus providing a new means by which remaining islet mass may be assessed in settings of diabetes. Generally, the method may provide a fast approach to provide new anatomical insight into pancreatic pathophysiology.

Place, publisher, year, edition, pages
Springer Nature, 2020
National Category
Cancer and Oncology
Identifiers
urn:nbn:se:umu:diva-180639 (URN)10.1038/s41598-020-74616-6 (DOI)000615370300001 ()33106532 (PubMedID)2-s2.0-85094218464 (Scopus ID)
Available from: 2021-02-25 Created: 2021-02-25 Last updated: 2022-09-15Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-5923-8572

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