Publications - Publikationer https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Bcontroller%5D=Publications&cHash=b93f275eb7f222ee054fa5e6f3c5acc7 en-us PURE Extension typo3support@science.au.dk (Web Department) 30 <![CDATA[Differential regulation of the interferon induced gene ISG12A by serum from healthy and preeclamptic pregnancies]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d491ecb8-8310-4cfd-bfe5-6dcff9c511bb&tx_pure_pure5%5BshowType%5D=pub&cHash=fb75b5a6530c81f17777f62eb49f94f0 Kronborg, C. S., Knudsen, U. B., Martensen, P. M. Endothelial-cell dysfunction is central in the preeclamptic pathogenesis. Several components present in the blood of the preeclamptic mother are capable of mediating this dysfunction. We analyzed the regulation of the ISG12A gene by serum from the third trimester, to elucidate the role of type 1 interferon ISGs late in both healthy and preeclamptic pregnancies. The ISG12A transcription was up-regulated by serum from healthy pregnant women, but not by preeclamptic serum in HeLa and human umbilical vein endothelial cells (HUVEC). However, the ISG12A up-regulation by healthy pregnancy serum was not due to a general type 1 interferon response, since 6-16 and OAS1 were not up-regulated similarly. Also, the up-regulation of ISG12A was independent of the interferon-α receptor 2, but dependent on STAT1. Stimulation with folic acid alone or in combination with preeclamptic serum up-regulated ISG12A and 6-16. We conclude that type 1 interferon is not increased in third trimester serum, neither from healthy nor preeclamptic pregnancies. However, since ISG12A mRNA is up-regulated in healthy pregnancies, the ISG12A protein might take part in maintaining endothelial stability, as this function is lacking in preeclamptic pregnancies. Folic acid may ameliorate endothelial cell stability in preeclampsia by up-regulating ISG12A.

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Forskning Tue, 01 Apr 2008 09:07:34 +0200 d491ecb8-8310-4cfd-bfe5-6dcff9c511bb
<![CDATA[Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=829b7c27-aa62-4bef-a5a0-f56b980fd7db&tx_pure_pure5%5BshowType%5D=pub&cHash=a8258fa21659e9b43d074150a5f4b718 Hansen, M. F., Greibe, E., Skovbjerg, S., et al. The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells.We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR.The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

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Forskning Wed, 01 Jul 2015 09:07:34 +0200 829b7c27-aa62-4bef-a5a0-f56b980fd7db
<![CDATA[A robust immunoassay for pregnancy-associated plasma protein-A2 based on analysis of circulating antigen]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=7742abcc-1696-4631-b6c4-c57af6d89278&tx_pure_pure5%5BshowType%5D=pub&cHash=5e858f5ba16f1643dac0cde539ea01eb Kloverpris, S., Gaidamauskas, E., Rasmussen, L. C.V., et al. Pregnancy-associated plasma protein-A (PAPP-A) and PAPP-A2, two homologous metzincin metalloproteases, are both tightly linked to regulation within the insulin-like growth factor (IGF) system because of their specific cleavage of IGF binding proteins. Recent studies suggest that PAPP-A may be involved in clinical conditions related to unwanted cellular growth, and the circulating levels of PAPP-A is an established biomarker in prenatal screening for chromosomal abnormalities. Microarray data indicate that PAPP-A2 has potential as a biomarker for pre-eclampsia. However, well-characterized immunological methods of quantification are not available.We therefore developed monoclonal antibodies against recombinant PAPP-A2. The antibodies were epitope mapped against recombinantly expressed chimeras between PAPPA2 and PAPP-A. Furthermore, circulating PAPP-A2 was immunoaffinity purified and characterized by sequence analysis and mass spectrometry. Unlike PAPP-A, PAPP-A2 is a noncovalent dimer in which each subunit of 1558 amino acids originates fromall of the 22 predicted coding exons. A previously hypothesized variant (PAPP-E) does not exist, but low amounts of a C-terminally truncated PAPP-A2 variant was detected. A sensitive and robust ELISA for full-length PAPP-A2 was developed and used to establish normal ranges of PAPP-A2 through pregnancy.The functional sensitivity of this ELISA at 20% CV was 0.08 ng/ml, and the serum concentration of PAPP-A2 was found to increase during pregnancy in agreement with placental synthesis. The existence of this assay will enable an assessment of the biomarker potential of PAPP-A2 in pre-eclampsia as well as other clinical conditions.

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Forskning Fri, 01 Nov 2013 09:07:34 +0100 7742abcc-1696-4631-b6c4-c57af6d89278
<![CDATA[Differential regulation of the interferon induced gene ISG12A by serum from healthy and preeclamptic pregnancies]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c217beb7-f889-4016-a427-5f22e3c47871&tx_pure_pure5%5BshowType%5D=pub&cHash=e55c75b5d52c8b2aefee511968d5a46a Kronborg, C. S., Knudsen, U. B., Martensen, P. M. Endothelial-cell dysfunction is central in the preeclamptic pathogenesis. Several components present in the blood of the preeclamptic mother are capable of mediating this dysfunction. We analyzed the regulation of the ISG12A gene by serum from the third trimester, to elucidate the role of type 1 interferon ISGs late in both healthy and preeclamptic pregnancies. The ISG12A transcription was up-regulated by serum from healthy pregnant women, but not by preeclamptic serum in HeLa and human umbilical vein endothelial cells (HUVEC). However, the ISG12A up-regulation by healthy pregnancy serum was not due to a general type 1 interferon response, since 6-16 and OAS1 were not up-regulated similarly. Also, the up-regulation of ISG12A was independent of the interferon-alpha receptor 2, but dependent on STAT1. Stimulation with folic acid alone or in combination with preeclamptic serum up-regulated ISG12A and 6-16. We conclude that type 1 interferon is not increased in third trimester serum, neither from healthy nor preeclamptic pregnancies. However, since ISG12A mRNA is up-regulated in healthy pregnancies, the ISG12A protein might take part in maintaining endothelial stability, as this function is lacking in preeclamptic pregnancies. Folic acid may ameliorate endothelial cell stability in preeclampsia by up-regulating ISG12A.

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Forskning Tue, 01 Apr 2008 09:07:34 +0200 c217beb7-f889-4016-a427-5f22e3c47871
<![CDATA[Reduction of serum-induced endothelial STAT3(Y705) activation is associated with preeclampsia]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=fd6c55f3-87b6-4e92-84d0-716cce74aa3d&tx_pure_pure5%5BshowType%5D=pub&cHash=8b5deb0440fa07a339b2af481fd4ddab Christensen, M., Petersen, J. L., Sivanandam, P., Kronborg, C. S., Knudsen, U. B., Martensen, P. M. OBJECTIVES: Preeclampsia is associated with maternal morbidity and mortality during pregnancy, and also an increased cardiovascular disease (CVD) risk later in life. During preeclampsia, alterations in secreted placental factors leading to systemic maternal endothelial dysfunction are evident. However, little is known about the associated endothelial intracellular signaling. STAT3 is a latent cytoplasmic transcription factor involved in endothelial cell differentiation, survival, and angiogenesis. We aimed to test if preeclampsia and preeclampsia-related placental factors could alter serum-induced STAT3(Y705) activation in endothelial cells. Furthermore, if altered serum-induced endothelial STAT3 (Y705) activation is related to post-preeclamptic CVD risk.

STUDY DESIGN: HUVECs were used as a model of maternal endothelium. Experiments entailed addition of 20% human pregnancy serum as well as addition of recombinant PlGF, sFLT1 and VEGF-A165a to the cells.

MAIN OUTCOME MEASURES: Levels of pSTAT3(Y705) related to STAT3 levels were evaluated by immunoblotting analysis.

RESULTS: Our results show that preeclamptic serum induces significantly lower STAT3(Y705) phosphorylation compared with uncomplicated pregnancy serum (P = 0.0089) in endothelial cells. Furthermore, STAT3(Y705) phosphorylation was not changed upon addition of PlGF, sFLT1, or VEGF-A165a together with pregnancy sera compared with sera alone. Finally, sera from women with previous preeclampsia and current hypertension and carotid atherosclerotic plaques show significantly lower STAT3(Y705) phosphorylation capabilities compared with healthy women with previous uncomplicated pregnancies 8-18 years after deliveries (P = 0.029).

CONCLUSIONS: Reduction in serum-induced endothelial STAT3(Y705) activation may play an important role in the preeclampsia-associated endothelial dysfunction. Additionally, reduced endothelial STAT3(Y705) phosphorylation may contribute to increased post-preeclamptic CVD risk 8-18 years after delivery.

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Forskning Sun, 01 Aug 2021 09:07:34 +0200 fd6c55f3-87b6-4e92-84d0-716cce74aa3d
<![CDATA[Dietary intake of a MFGM/EV-rich concentrate promotes accretion of very long odd-chain sphingolipids and increases lipid metabolic turnover at the whole-body level]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=a10eaa80-5e9c-4d9b-ae8a-fe65d9a092b3&tx_pure_pure5%5BshowType%5D=pub&cHash=dd3b88dd0c9139e7096b75497075ecfa Sprenger, R. R., Bilgin, M., Ostenfeld, M. S., Bjørnshave, A., Rasmussen, J. T., Ejsing, C. S. Lipids from cow milk fat globule membranes (MFGMs) and extracellular vesicles (EVs) are considered beneficial for neurodevelopment, cognitive maintenance and human health in general. Nevertheless, it is largely unknown whether intake of infant formulas and medical nutrition products rich in these particles promote accretion of specific lipids and whether this affects metabolic homeostasis. To address this, we carried out a 16-week dietary intervention study where mice were supplemented with a MFGM/EV-rich concentrate, a control diet supplemented with a whey protein concentrate and devoid of milk lipids, or regular chow. Assessment of commonly used markers of metabolic health, including body weight, glucose intolerance and liver microanatomy, demonstrated no differences across the dietary regimes. In contrast, in-depth lipidomic analysis revealed accretion of milk-derived very long odd-chain sphingomyelins and ceramides in blood plasma and multiple tissues of mice fed the MFGM/EV diet. Furthermore, lipidomic flux analysis uncovered that mice fed the MFGM/EV diet have increased lipid metabolic turnover at the whole-body level. These findings help fill a long-lasting knowledge gap between the intake of MFGM/EV-containing foods and the health-promoting effects of their lipid constituents. In addition, the findings suggest that dietary sphingomyelins or ceramide-breakdown products with very long-chains can be used as structural components of cellular membranes, lipoprotein particles and signaling molecules that modulate metabolic homeostasis and health.

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Forskning Thu, 01 Aug 2024 09:07:34 +0200 a10eaa80-5e9c-4d9b-ae8a-fe65d9a092b3
<![CDATA[FastDFE]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=b40c875d-ea57-4a5d-9fcc-46ea3ef68e76&tx_pure_pure5%5BshowType%5D=pub&cHash=819d711602401c6b07f70338d136bd4d Sendrowski, J., Bataillon, T. Estimating the distribution of fitness effects (DFE) of new mutations is of fundamental importance in evolutionary biology, ecology, and conservation. However, existing methods for DFE estimation suffer from limitations, such as slow computation speed and limited scalability. To address these issues, we introduce fastDFE, a Python-based software package, offering fast, and flexible DFE inference from site-frequency spectrum (SFS) data. Apart from providing efficient joint inference of multiple DFEs that share parameters, it offers the feature of introducing genomic covariates that influence the DFEs and testing their significance. To further simplify usage, fastDFE is equipped with comprehensive VCF-To-SFS parsing utilities. These include options for site filtering and stratification, as well as site-degeneracy annotation and probabilistic ancestral-Allele inference. fastDFE thereby covers the entire workflow of DFE inference from the moment of acquiring a raw VCF file. Despite its Python foundation, fastDFE incorporates a full R interface, including native R visualization capabilities. The package is comprehensively tested and documented at fastdfe.readthedocs.io.

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Forskning Wed, 01 May 2024 09:07:34 +0200 b40c875d-ea57-4a5d-9fcc-46ea3ef68e76
<![CDATA[CryoEM reconstructions of membrane proteins solved in several amphipathic solvents, nanodisc, amphipol and detergents, yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c2ed2561-c54e-4c26-afca-7da69cd2f54b&tx_pure_pure5%5BshowType%5D=pub&cHash=c984ebe48f8dae9ef164dad76691e8e2 Zampieri, V., Gobet, A., Robert, X., Falson, P., Chaptal, V. To maintain membrane proteins soluble in aqueous solution, amphipathic compounds are used to shield the hydrophobic patch of their membrane insertion, which forms a belt around the protein. This amphipathic belt is seldom looked at due to the difficulty to visualize it. Cryo-EM is now offering this possibility, where belts are visible in 3D reconstructions. We investigated membrane proteins solved in nanodiscs, amphipols or detergents to analyze whether the nature of the amphipathic compound influences the belt size in 3D reconstructions. We identified belt boundaries in map-density distributions and measured distances for every reconstruction. We showed that all the belts create on average similar reconstructions, whether they originate from the same protein, or from protein from different shapes and structures. There is no difference among detergents or types of nanodisc used. These observations illustrate that the belt observed in 3D reconstructions corresponds to the minimum ordered layer around membrane proteins.

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Forskning Mon, 01 Nov 2021 09:07:34 +0100 c2ed2561-c54e-4c26-afca-7da69cd2f54b
<![CDATA[Substrate-bound and substrate-free outward-facing structures of a multidrug ABC exporter]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=95a34726-60c9-4220-901d-02cea9c79cf5&tx_pure_pure5%5BshowType%5D=pub&cHash=5c7748a6a7173875a3a06406ffbbb528 Chaptal, V., Zampieri, V., Wiseman, B., et al. Multidrug ABC transporters translocate drugs across membranes by a mechanism for which the molecular features of drug release are so far unknown. Here, we resolved three ATP-Mg2+-bound outward-facing conformations of the Bacillus subtilis (homodimeric) BmrA by x-ray crystallography and single-particle cryo-electron microscopy (EM) in detergent solution, one of them with rhodamine 6G (R6G), a substrate exported by BmrA when overexpressed in B. subtilis. Two R6G molecules bind to the drug-binding cavity at the level of the outer leaflet, between transmembrane (TM) helices 1-2 of one monomer and TM5'-6' of the other. They induce a rearrangement of TM1-2, highlighting a local flexibility that we confirmed by hydrogen/deuterium exchange and molecular dynamics simulations. In the absence of R6G, simulations show a fast postrelease occlusion of the cavity driven by hydrophobicity, while when present, R6G can move within the cavity, maintaining it open.

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Forskning Fri, 28 Jan 2022 09:07:34 +0100 95a34726-60c9-4220-901d-02cea9c79cf5
<![CDATA[Overexpression of the ABC Transporter BmrA Within Intracellular Caveolae in Escherichia coli]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=405f1404-d611-4c81-bcc9-1e9149ec471d&tx_pure_pure5%5BshowType%5D=pub&cHash=7339df67e44596f5147e95cb868af7b6 Gobet, A., Jaxel, C., Magnard, S., et al. We describe here the overproduction and oriented membrane insertion of membrane protein inside intracellular vesicles named heterologous caveolae within E. coli. The method is described with BmrA, a multidrug efflux pump from Bacillus subtilis. BmrA is produced in these vesicles, thanks to the coexpression with the canine caveolin-1β, one of the two isoforms of caveolin-1. Enriched by sucrose gradient, the caveolae-containing fraction allows to probe the ATPase and Hoechst 33342 transport activities, the latter displaying a higher specific activity than the same without caveolin-1β.

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Forskning Sat, 01 Jan 2022 09:07:34 +0100 405f1404-d611-4c81-bcc9-1e9149ec471d
<![CDATA[The non-Newtonian behavior of detergents during concentration is increased by macromolecules, in trans, and results in their over-concentration]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d0755d71-0705-455e-b1e0-480c8472e589&tx_pure_pure5%5BshowType%5D=pub&cHash=1c473bd354c3b8d39f224229da7ef701 Gobet, A., Zampieri, V., Magnard, S., Pebay-Peyroula, E., Falson, P., Chaptal, V. Concentration of pure membrane proteins in detergent solution results in detergent concentration, albeit in unknown amounts. This phenomenon is observed in every lab working on membrane proteins, but has seldom been investigated. In this study, we explored the behavior of detergents mixed with membrane proteins during the step of sample concentration using centrifugal devices. We show that detergent over-concentrate with the presence of polymers, typically membrane or soluble proteins but also polysaccharides. The over-concentration of detergents depends on centrifugal force applied to the device. With the use of a specific dye, we observed the formation of a mesh on the concentrator device. Importantly, reducing the centrifugal speed allows to reduce the concentration of detergents when mixed to macromolecules, as tested with 3 different membrane proteins. All together, these results highlight the non-Newtonian behavior of detergents and provides a solid framework to investigators to improve drastically biochemical and structural studies of membrane proteins.

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Forskning Wed, 01 Feb 2023 09:07:34 +0100 d0755d71-0705-455e-b1e0-480c8472e589
<![CDATA[Conformational space exploration of cryo-EM structures by variability refinement]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=6f9f5740-b93f-4560-be5e-d5d3df7c76ad&tx_pure_pure5%5BshowType%5D=pub&cHash=457869830ae9d656b228146971795e5c Afonine, P. V., Gobet, A., Moissonnier, L., Martin, J., Poon, B. K., Chaptal, V. Cryo-EM observation of biological samples enables visualization of sample heterogeneity, in the form of discrete states that are separable, or continuous heterogeneity as a result of local protein motion before flash freezing. Variability analysis of this continuous heterogeneity describes the variance between a particle stack and a volume, and results in a map series describing the various steps undertaken by the sample in the particle stack. While this observation is absolutely stunning, it is very hard to pinpoint structural details to elements of the maps. In order to bridge the gap between observation and explanation, we designed a tool that refines an ensemble of structures into all the maps from variability analysis. Using this bundle of structures, it is easy to spot variable parts of the structure, as well as the parts that are not moving. Comparison with molecular dynamics simulations highlights the fact that the movements follow the same directions, albeit with different amplitudes. Ligand can also be investigated using this method. Variability refinement is available in the Phenix software suite, accessible under the program name phenix.varref.

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Forskning Sat, 01 Apr 2023 09:07:34 +0200 6f9f5740-b93f-4560-be5e-d5d3df7c76ad
<![CDATA[CryoEM Data Analysis of Membrane Proteins. Practical Considerations on Amphipathic Belts, Ligands, and Variability Analysis]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=28c5176a-3572-42f9-bd0c-684c2385c5dd&tx_pure_pure5%5BshowType%5D=pub&cHash=72abe1bcf0858a4db5cc167f83b5e210 Gobet, A., Moissonnier, L., Chaptal, V. Membrane proteins data analysis by cryoEM shows some specificities, as can be found in other typical investigations such as biochemistry, biophysics, or X-ray crystallography. Membrane proteins are typically surrounded by an amphipathic belt that will have some degree of influence on the 3D reconstruction and analysis. In this chapter, we review our experience with the ABC transporter BmrA, as well as our statistical analysis of amphipathic belts around membrane proteins, to bring awareness on some particular features of membrane protein investigations by cryoEM.

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Forskning Mon, 01 Jan 2024 09:07:34 +0100 28c5176a-3572-42f9-bd0c-684c2385c5dd
<![CDATA[The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=499953a9-fad7-4e6f-ba2a-bbad04984676&tx_pure_pure5%5BshowType%5D=pub&cHash=c375a231471da526a6a73f8e7420e789 Di Cesare, M., Kaplan, E., Rendon, J., et al. ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

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Forskning Mon, 01 Jan 2024 09:07:34 +0100 499953a9-fad7-4e6f-ba2a-bbad04984676
<![CDATA[Feed intake in housed dairy cows]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=a4684126-c7aa-4791-ae59-6b8db94ba4da&tx_pure_pure5%5BshowType%5D=pub&cHash=d186a5eb03434d99c9d9ef37c5cfb5aa Giagnoni, G., Lassen, J., Lund, P., Foldager, L., Johansen, M., Weisbjerg, M. R. Measuring feed intake accurately is crucial to determine feed efficiency and for genetic selection. A system using three-dimensional (3D) cameras and deep learning algorithms can measure the volume of feed intake in dairy cows, but for now, the system has not been validated for feed intake expressed as weight of feed. The aim of this study was to validate the weight of feed intake predicted from the 3D cameras with the actual measured weight. It was hypothesised that diet−specific coefficients are necessary for predicting changes in weight, that the relationship between weight and volume is curvilinear throughout the day, and that manually pushing the feed affects this relationship. Twenty-four lactating Danish Holstein cows were used in a cross-over design with four dietary treatments, 2 × 2 factorial arranged with either grass-clover silage or maize silage as silage factor, and barley or dried beet pulp as concentrate factor. Cows were adapted to the diets for 11 d, and for 3 d to tie-stall housing before camera measurements. Six cameras were used for recording, each mounted over an individual feeding platform equipped with a weight scale. When building the predictive models, four cameras were used for training, and the remaining two for testing the prediction of the models. The most accurate predictions were found for the average feed intake over a period when using the starting density of the feed pile, which resulted in the lowest errors, 6% when expressed as RMSE and 5% expressed as mean absolute error. A model including curvilinear effects of feed volume and the impact of manual feed pushing was used on a dataset including daily time points. When cross-validating, the inclusion of a curvilinear effect and a feed push effect did not improve the accuracy of the model for neither the feed pile nor the feed removed by the cow between consecutive time points. In conclusion, measuring daily feed intake from this 3D camera system in the present experimental setup could be accomplished with an acceptable error (below 8%), but the system should be improved for individual meal intake measurements if these measures were to be implemented.

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Forskning Sat, 01 Jun 2024 09:07:34 +0200 a4684126-c7aa-4791-ae59-6b8db94ba4da
<![CDATA[Mining and engineering activity in catalytic amyloids]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=8843b42e-f8aa-4ac6-bd39-078baef4a0cd&tx_pure_pure5%5BshowType%5D=pub&cHash=5be4ce1cded8104730f8629e9186e227 Peña-Díaz, S., Ferreira, P., Ramos, M. J., Otzen, D. E. This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.

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Forskning Mon, 01 Jan 2024 09:07:34 +0100 8843b42e-f8aa-4ac6-bd39-078baef4a0cd
<![CDATA[Gene expression responses to FUS, EWS, and TAF15 reduction and stress granule sequestration analyses identifies FET-protein non-redundant functions]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=bd62c4a1-79fb-4ebe-a400-6e246c63f5de&tx_pure_pure5%5BshowType%5D=pub&cHash=70bcdbba48d4a80959978499961177fb Blechingberg, J., Luo, Y., Bolund, L., Damgaard, C. K., Nielsen, A. L. The FET family of proteins is composed of FUS/TLS, EWS/EWSR1, and TAF15 and possesses RNA- and DNA-binding capacities. The FET-proteins are involved in transcriptional regulation and RNA processing, and FET-gene deregulation is associated with development of cancer and protein granule formations in amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and trinucleotide repeat expansion diseases. We here describe a comparative characterization of FET-protein localization and gene regulatory functions. We show that FUS and TAF15 locate to cellular stress granules to a larger extend than EWS. FET-proteins have no major importance for stress granule formation and cellular stress responses, indicating that FET-protein stress granule association most likely is a downstream response to cellular stress. Gene expression analyses showed that the cellular response towards FUS and TAF15 reduction is relatively similar whereas EWS reduction resulted in a more unique response. The presented data support that FUS and TAF15 are more functionally related to each other, and that the FET-proteins have distinct functions in cellular signaling pathways which could have implications for the neurological disease pathogenesis.

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Forskning Sun, 01 Jan 2012 09:07:34 +0100 bd62c4a1-79fb-4ebe-a400-6e246c63f5de
<![CDATA[A 5' splice site enhances the recruitment of basal transcription initiation factors in vivo]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=5ef98eef-c97e-43c3-afa1-dea803a7505d&tx_pure_pure5%5BshowType%5D=pub&cHash=584713370b6239542cd49301eeb927e4 Damgaard, C. K., Kahns, S., Lykke-Andersen, S., Nielsen, A. L., Jensen, T. H., Kjems, J. Transcription and pre-mRNA splicing are interdependent events. Although mechanisms governing the effects of transcription on splicing are becoming increasingly clear, the means by which splicing affects transcription remain elusive. Using cell lines stably expressing HIV-1 or beta-globin mRNAs, harboring wild-type or various 5' splice site mutations, we demonstrate a strong positive correlation between splicing efficiency and transcription activity. Interestingly, a 5' splice site can stimulate transcription even in the absence of splicing. Chromatin immunoprecipitation experiments show enhanced promoter docking of transcription initiation factors TFIID, TFIIB, and TFIIH on a gene containing a functional 5' splice site. In addition to their promoter association, the TFIID and TFIIH components, TBP and p89, are specifically recruited to the 5' splice site region. Our data suggest a model in which a promoter-proximal 5' splice site via its U1 snRNA interaction can feed back to stimulate transcription initiation by enhancing pre-initiation complex assembly.

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Forskning Fri, 01 Feb 2008 09:07:34 +0100 5ef98eef-c97e-43c3-afa1-dea803a7505d
<![CDATA[Neuroectoderm phenotypes in a human stem cell model of O-GlcNAc transferase associated with intellectual disability]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=dbcd2847-56d0-4ba8-b6a9-d36d25d949a2&tx_pure_pure5%5BshowType%5D=pub&cHash=9abe5488eba9d086a25bd2955beae61d Murray, M., Davidson, L., Ferenbach, A. T., Lefeber, D., van Aalten, D. M.F. Pathogenic variants in the O-GlcNAc transferase gene (OGT) have been associated with a congenital disorder of glycosylation (OGT-CDG), presenting with intellectual disability which may be of neuroectodermal origin. To test the hypothesis that pathology is linked to defects in differentiation during early embryogenesis, we developed an OGT-CDG induced pluripotent stem cell line together with isogenic control generated by CRISPR/Cas9 gene-editing. Although the OGT-CDG variant leads to a significant decrease in OGT and O-GlcNAcase protein levels, there were no changes in differentiation potential or stemness. However, differentiation into ectoderm resulted in significant differences in O-GlcNAc homeostasis. Further differentiation to neuronal stem cells revealed differences in morphology between patient and control lines, accompanied by disruption of the O-GlcNAc pathway. This suggests a critical role for O-GlcNAcylation in early neuroectoderm architecture, with robust compensatory mechanisms in the earliest stages of stem cell differentiation.

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Forskning Sat, 01 Jun 2024 09:07:34 +0200 dbcd2847-56d0-4ba8-b6a9-d36d25d949a2
<![CDATA[Automatic removal of soft tissue from 3D dental photo scans; an important step in automating future forensic odontology identification]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=729bd59d-157f-4e00-991d-176dac179ae4&tx_pure_pure5%5BshowType%5D=pub&cHash=b307f81dbae6319b841dbb713ef316ae Kofod Petersen, A., Forgie, . A. H., Bindslev, D. A., Villesen, P., Staun Larsen, L. Forskning Wed, 01 May 2024 09:07:34 +0200 729bd59d-157f-4e00-991d-176dac179ae4 <![CDATA[Population genomics of the muskox' resilience in the near absence of genetic variation]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=b7ce4b55-54e5-4b24-a20e-98d6c2a603fd&tx_pure_pure5%5BshowType%5D=pub&cHash=f54103e87a67bdeaa561e6b63094397f Pečnerová, P., Lord, E., Garcia-Erill, G., et al. Genomic studies of species threatened by extinction are providing crucial information about evolutionary mechanisms and genetic consequences of population declines and bottlenecks. However, to understand how species avoid the extinction vortex, insights can be drawn by studying species that thrive despite past declines. Here, we studied the population genomics of the muskox (Ovibos moschatus), an Ice Age relict that was at the brink of extinction for thousands of years at the end of the Pleistocene yet appears to be thriving today. We analysed 108 whole genomes, including present-day individuals representing the current native range of both muskox subspecies, the white-faced and the barren-ground muskox (O. moschatus wardi and O. moschatus moschatus) and a ~21,000-year-old ancient individual from Siberia. We found that the muskox' demographic history was profoundly shaped by past climate changes and post-glacial re-colonizations. In particular, the white-faced muskox has the lowest genome-wide heterozygosity recorded in an ungulate. Yet, there is no evidence of inbreeding depression in native muskox populations. We hypothesize that this can be explained by the effect of long-term gradual population declines that allowed for purging of strongly deleterious mutations. This study provides insights into how species with a history of population bottlenecks, small population sizes and low genetic diversity survive against all odds.

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Forskning Mon, 01 Jan 2024 09:07:34 +0100 b7ce4b55-54e5-4b24-a20e-98d6c2a603fd
<![CDATA[A Multi-Faceted Binding Assessment of Aptamers Targeting the SARS-CoV-2 Spike Protein]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=556132b1-92df-4d9b-b66f-d40299a5baaf&tx_pure_pure5%5BshowType%5D=pub&cHash=91548f4f6f67117142c8d524ac7327f3 Civit, L., Moradzadeh, N., Jonczyk, A., et al. The COVID-19 pandemic has underscored the critical need for the advancement of diagnostic and therapeutic platforms. These platforms rely on the rapid development of molecular binders that should facilitate surveillance and swift intervention against viral infections. In this study, we have evaluated by three independent research groups the binding characteristics of various published RNA and DNA aptamers targeting the spike protein of the SARS-CoV-2 virus. For this comparative analysis, we have employed different techniques such as biolayer interferometry (BLI), enzyme-linked oligonucleotide assay (ELONA), and flow cytometry. Our data show discrepancies in the reported specificity and affinity among several of the published aptamers and underline the importance of standardized methods, the impact of biophysical techniques, and the controls used for aptamer characterization. We expect our results to contribute to the selection and application of suitable aptamers for the detection of SARS-CoV-2.

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Forskning Wed, 01 May 2024 09:07:34 +0200 556132b1-92df-4d9b-b66f-d40299a5baaf
<![CDATA[A phosphate transporter in VIPergic neurons of the suprachiasmatic nucleus gates locomotor activity during the light/dark transition in mice]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=f44ff5a7-4016-45eb-82cf-01bb32085340&tx_pure_pure5%5BshowType%5D=pub&cHash=71760f28a97af87f710b1adac539b859 Pierre-Ferrer, S., Collins, B., Lukacsovich, D., et al. The suprachiasmatic nucleus (SCN) encodes time of day through changes in daily firing; however, the molecular mechanisms by which the SCN times behavior are not fully understood. To identify factors that could encode day/night differences in activity, we combine patch-clamp recordings and single-cell sequencing of individual SCN neurons in mice. We identify PiT2, a phosphate transporter, as being upregulated in a population of Vip+Nms+ SCN neurons at night. Although nocturnal and typically showing a peak of activity at lights off, mice lacking PiT2 (PiT2−/−) do not reach the activity level seen in wild-type mice during the light/dark transition. PiT2 loss leads to increased SCN neuronal firing and broad changes in SCN protein phosphorylation. PiT2−/− mice display a deficit in seasonal entrainment when moving from a simulated short summer to longer winter nights. This suggests that PiT2 is responsible for timing activity and is a driver of SCN plasticity allowing seasonal entrainment.

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Forskning Wed, 01 May 2024 09:07:34 +0200 f44ff5a7-4016-45eb-82cf-01bb32085340
<![CDATA[A high fat to vitamin E ratio in the feed protects and improves uptake of the natural form of vitamin E in postweaning calves]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=51ad5c57-19ea-4880-b423-9ce2708c6254&tx_pure_pure5%5BshowType%5D=pub&cHash=d896bb9ad0ca7f04ebdb38322cc52dcf Lashkari, S., Jensen, S. K., Foldager, L., Larsen, T., Vestergaard, M. . In postweaning calves, it is a challenge to maintain the plasma vitamin E level at or above the recommended level (3 µg/mL), which is linked to a good immune response. It has been unclear until now why the provision of solid feed with concentrations below 200 mg/kg feed of vitamin E is ineffective in maintaining the plasma vitamin E level of calves above the recommended plasma level postweaning. The present study was conducted to investigate if a high fat to vitamin E ratio in the concentrate could protect and improve the delivery of the natural form of vitamin E (RRR-α-tocopherol) to calves postweaning. Thirty calves were included in the experiment from 2 weeks preweaning until 2 weeks postweaning (Weeks −2, −1, 0 [weaning], 1, and 2 relative to weaning) and fed one of three concentrates in which lecithin mixture provided the fat supplement: control (77 mg/kg of vitamin E and 4.9% DM of crude fat; CONT), medium level of vitamin E supplemented (147 mg/kg of vitamin E and 7.7% DM of crude fat; MedVE) or high level of vitamin E supplemented (238 mg/kg of vitamin E and 12.4% DM of fat; HiVE). Thus, there was a comparable ratio of fat to vitamin E (520–630) in the three concentrates. During the 2 weeks postweaning, final body weight (92 ± 2 kg), average daily gain (917 ± 51 g/day) and concentrate intake (2.2 ± 0.09 kg/day; mean of treatment ± standard error) were unaffected by treatment and the interaction between treatment and week. There was an interaction between treatment and week for vitamin E intake pre- (p < 0.001) and postweaning (p < 0.001). There was an interaction between treatment and week (p < 0.001) for plasma vitamin E level postweaning, and it was 2.5, 3.1, and 3.8 µg/mL in CONT, MedVE, and HiVE, respectively, at Week 1 postweaning. In addition, plasma vitamin E levels at Week 2 postweaning were 2.6, 3.6 and 4.8 µg/mL in CONT, MidVE and HiVE respectively. The results show that 147 mg/kg of lecithin-protected vitamin E in the concentrate is needed to secure a plasma vitamin E level well above the recommended level. In addition, lecithin-protected vitamin E elevated the plasma level of triglycerides and nonesterified fatty acids.

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Forskning Wed, 01 May 2024 09:07:34 +0200 51ad5c57-19ea-4880-b423-9ce2708c6254
<![CDATA[Do milk proteins relieve capsaicin-induced burning sensation in the oral cavity?]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=47ffa8d9-917a-42e6-a50c-cf868abb0d34&tx_pure_pure5%5BshowType%5D=pub&cHash=4aad23bc80bddee5c56353211634cf7d Gøkhan, M. A., Sørensen, E. S., Baad-Hansen, L. Milk has a soothing effect on the capsaicin-induced burning sensation in the mouth. This double-blinded, placebo-controlled, cross-over study aimed to investigate if milk proteins could relieve the capsaicin-induced burning sensation. During each session, the tongue of each participant was exposed to capsaicin twice for 8 min in total. Subsequently, the participants rinsed the mouth with one of three solutions: 5% casein, 5% whey protein, or water. The participants rated the perceived unpleasantness and burning sensation during capsaicin exposure and after rinsing on numerical rating scales. Thermographic imaging and semi-quantitative sensory testing were performed at baseline, after capsaicin exposure, and after rinsing. No significant differences were observed between sessions in any of the measured parameters (p ≥.053). Scores for unpleasantness and burning sensation varied over time (p ≤.006). Heat and mechanical sensitivity changed over time (p <.001). In conclusion, rinsing with milk protein solutions did not have any robust effect in this study design. Practical Applications: It has been suggested that milk proteins could be responsible for the soothing effect milk has on the capsaicin-induced burning sensation in the mouth. The present results indicate that milk proteins are not solely responsible for this effect. Shedding light on the effects milk and its constituents have on the oral mucosa during a state of burning sensation or pain is of importance. This can help determining if specific constituents of milk could be used to relieve meal-related burning sensations as well as illness-related acute or chronic pain in the oral mucosa.

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Forskning Sat, 01 Jun 2024 09:07:34 +0200 47ffa8d9-917a-42e6-a50c-cf868abb0d34
<![CDATA[Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=5996b194-9c40-4cb2-b76d-413f46b8dd6c&tx_pure_pure5%5BshowType%5D=pub&cHash=4e7114f524bcf25ee076521b08a735da Singh, V., Itoh, Y., Del'Olio, S., et al. Forskning Mon, 20 May 2024 09:07:34 +0200 5996b194-9c40-4cb2-b76d-413f46b8dd6c <![CDATA[Neurodevelopmental defects in a mouse model of O-GlcNAc transferase intellectual disability]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=16b547c1-4f94-4258-8dbb-e205a50ac255&tx_pure_pure5%5BshowType%5D=pub&cHash=f60f6f4a0282069d3bdcafe4d5db7aff Authier, F., Ondruskova, N., Ferenbach, A. T., McNeilly, A. D., van Aalten, D. M.F. The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to proteins (referred to as O-GlcNAcylation) is a modification that is crucial for vertebrate development. O-GlcNAcylation is catalyzed by O-GlcNAc transferase (OGT) and reversed by O-GlcNAcase (OGA). Missense variants of OGT have recently been shown to segregate with an X-linked syndromic form of intellectual disability, OGT-linked congenital disorder of glycosylation (OGT-CDG). Although the existence of OGT-CDG suggests that O-GlcNAcylation is crucial for neurodevelopment and/or cognitive function, the underlying pathophysiologic mechanisms remain unknown. Here we report a mouse line that carries a catalytically impaired OGT-CDG variant. These mice show altered O-GlcNAc homeostasis with decreased global O-GlcNAcylation and reduced levels of OGT and OGA in the brain. Phenotypic characterization of the mice revealed lower body weight associated with reduced body fat mass, short stature and microcephaly. This mouse model will serve as an important tool to study genotype-phenotype correlations in OGT-CDG in vivo and for the development of possible treatment avenues for this disorder.

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Forskning Mon, 01 Apr 2024 09:07:34 +0200 16b547c1-4f94-4258-8dbb-e205a50ac255
<![CDATA[Error-Corrected Deep Targeted Sequencing of Circulating Cell-Free DNA from Colorectal Cancer Patients for Sensitive Detection of Circulating Tumor DNA]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=851e94a0-171c-4621-9aa9-d072537a1dcd&tx_pure_pure5%5BshowType%5D=pub&cHash=22a7dcab6166dfea32b0bf2c83515d79 Frydendahl, A., Rasmussen, M. H., Jensen, S. Ø., et al. Circulating tumor DNA (ctDNA) is a promising biomarker, reflecting the presence of tumor cells. Sequencing-based detection of ctDNA at low tumor fractions is challenging due to the crude error rate of sequencing. To mitigate this challenge, we developed ultra-deep mutation-integrated sequencing (UMIseq), a fixed-panel deep targeted sequencing approach, which is universally applicable to all colorectal cancer (CRC) patients. UMIseq features UMI-mediated error correction, the exclusion of mutations related to clonal hematopoiesis, a panel of normal samples for error modeling, and signal integration from single-nucleotide variations, insertions, deletions, and phased mutations. UMIseq was trained and independently validated on pre-operative (pre-OP) plasma from CRC patients (n = 364) and healthy individuals (n = 61). UMIseq displayed an area under the curve surpassing 0.95 for allele frequencies (AFs) down to 0.05%. In the training cohort, the pre-OP detection rate reached 80% at 95% specificity, while it was 70% in the validation cohort. UMIseq enabled the detection of AFs down to 0.004%. To assess the potential for detection of residual disease, 26 post-operative plasma samples from stage III CRC patients were analyzed. From this we found that the detection of ctDNA was associated with recurrence. In conclusion, UMIseq demonstrated robust performance with high sensitivity and specificity, enabling the detection of ctDNA at low allele frequencies.

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Forskning Mon, 01 Apr 2024 09:07:34 +0200 851e94a0-171c-4621-9aa9-d072537a1dcd
<![CDATA[Identificación de genes clave implicados en el síndrome de Down mediante terapia génica]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=2c7371fb-1dde-41ef-bbc9-8c88cf48f8dc&tx_pure_pure5%5BshowType%5D=pub&cHash=a65204881eb28feb9eb891af3816c9fe Fillat, C., Bofill-De Ros, X., Santos, M., et al. Viruses have evolved ways of encapsulating and delivering their genes into human cells. Gene therapy takes advantage of this capability to manipulate the viral genome and convert an infectious agent into an effcient vector that delivers therapeutic genes. In the current work we have applied gene therapy approaches based on adeno-associated virus and lentivirus delivery to identify candidate genes (protein-coding or miRNAs) involved in the cognitive defcits in Down Syndrome. We show that the hippocampal injection of the adeno-associated virus AAV2/1-shDyrk1A normalized Dyrk1A expression in the trisomic Ts65Dn mice. As a consequence the regulation of key molecular players in memory and learning processes was rescued and mice showed an attenuation of synaptic plasticity defects and improved effcacy in learning strategies. All together these results reinforce the role of Dyrk1A in cognition. On the other hand, with the lentiviral strategydeveloped to specifcally inhibit miR-155 and miR-802 (Lv-anti-miR155/802), we were able to show a tight control of the miRNAs target Mecp2 suggesting that the downregulation of MeCP2 in Down syndromecould be a contributing factor to the cognitive defects.

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Forskning Fri, 01 Aug 2014 09:07:34 +0200 2c7371fb-1dde-41ef-bbc9-8c88cf48f8dc
<![CDATA[Controlling adenoviral replication to induce oncolytic efficacy]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=d934cdd6-4b7c-4eae-8223-81e354cd14fa&tx_pure_pure5%5BshowType%5D=pub&cHash=eb4d5fba9d06c8dbe190ebdc1bbc84e3 Fillat, C., José, A., Bofill-de Ros, X., Mato-Berciano, A., Maliandi, M. V., Abate-Daga, D. Over the last decade, cancer therapy has found itself challenged by the growing field of oncolytic virotherapy.Many different viruses are currently under study, investigating their potential to induce antitumor effects through repeated cycles of viral infection and cell lysis. It was, however, genetically-engineered replication-selective adenoviruses that were the first to enter clinical trials with cancer patients. The difficulties involved in combining selectivity and elevated potency in a single oncolytic adenovirus have led investigators to design and test many different approaches. Different strategies, based on the control of viral replication, are presented in the current review. We discuss how the growing knowledge of cell and tumour biology, with the advances made in adenoviral virology, has inspired the fine-tuning of genetically-engineered adenoviruses Special emphasis is placed on the fundamentals behind the use of certain specific genetic elements, introduced into the viral genome to control viral gene expression and on describing the most important viral gene mutations.

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Forskning Fri, 01 Jan 2010 09:07:34 +0100 d934cdd6-4b7c-4eae-8223-81e354cd14fa
<![CDATA[IsomiRs]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=4da38148-0238-4592-a662-66eda523a29c&tx_pure_pure5%5BshowType%5D=pub&cHash=d827456cd5457504caf0426840b52085 Bofill-De Ros, X., Yang, A., Gu, S. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play increasingly appreciated roles in gene regulation. In animals, miRNAs silence gene expression by binding to partially complementary sequences within target mRNAs. It is well-established that miRNAs recognize canonical target sites by base-pairing in the 5′region. However, the development of biochemical methods has identified many novel, non-canonical target sites, suggesting additional modes of miRNA-target association. Here, we review the current knowledge of miRNA-target recognition and how new evidence supports or challenges existing models. We also review the process by which microRNA isoforms achieve functional diversification via modulation of target recognition.

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Forskning Wed, 01 Apr 2020 09:07:34 +0200 4da38148-0238-4592-a662-66eda523a29c
<![CDATA[Genome-wide miR-155 and miR-802 target gene identification in the hippocampus of Ts65Dn Down syndrome mouse model by miRNA sponges]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=436e8a55-6efa-4ebc-9924-531cd5287a97&tx_pure_pure5%5BshowType%5D=pub&cHash=693abe18fc2b46c3c4636b0e85334834 Bofill-De Ros, X., Santos, M., Vila-Casadesús, M., et al. Background: Down syndrome (DS) or trisomy 21 is the result of a genetic dosage imbalance that translates in a broad clinical spectrum. A major challenge in the study of DS is the identification of functional genetic elements with wide impact on phenotypic alterations. Recently, miRNAs have been recognized as major contributors to several disease conditions by acting as post-transcriptional regulators of a plethora of genes. Five chromosome 21 (HSA21) miRNAs have been found overexpressed in DS individuals and could function as key elements in the pathophysiology. Interestingly, in the trisomic Ts65Dn DS mouse model two of these miRNAs (miR-155 and miR-802) are also triplicated and overexpressed in brain. Results: In the current work, we interrogated the impact of miR-155 and miR-802 upregulation on the transcriptome of Ts65Dn brains. We developed a lentiviral miRNA-sponge strategy (Lv-miR155-802T) to identify in vivo relevant miR-155 and miR-802 target mRNAs. Hippocampal injections of lentiviral sponges in Ts65Dn mice normalized the expression of miR-155 and miR-802 and rescued the levels of their targets methyl-CpG-binding protein 2 gene (Mecp2), SH2 (Src homology 2)-containing inositol phosphatase-1 (Ship1) and Forkhead box protein M1 (FoxM1). Transcriptomic data of Lv-miR155-802T miRNA-sponge treated hippocampi correlated with candidate targets highlighting miRNA dosage-sensitive genes. Significant associations were found in a subset of genes (Rufy2, Nova1, Nav1, Thoc1 and Sumo3) that could be experimentally validated. Conclusions: The lentiviral miRNA-sponge strategy demonstrated the genome-wide regulatory effects of miR-155 and miR-802. Furthermore, the analysis combining predicted candidates and experimental transcriptomic data proved to retrieve genes with potential significance in DS-hippocampal phenotype bridging with DS other neurological-associated diseases such as Alzheimer's disease.

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Forskning Fri, 06 Nov 2015 09:07:34 +0100 436e8a55-6efa-4ebc-9924-531cd5287a97
<![CDATA[Flexible pri-miRNA structures enable tunable production of 5’ isomiRs]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=e32b92ec-c523-4454-807a-e1066ee7ade7&tx_pure_pure5%5BshowType%5D=pub&cHash=81a35a3635e0178da65deab04247b948 Bofill-De Ros, X., Hong, Z., Birkenfeld, B., et al. The Drosha cleavage of a pri-miRNA defines mature microRNA sequence. Drosha cleavage at alternative positions generates 5’ isoforms (isomiRs) which have distinctive functions. To understand how pri-miRNA structures influence Drosha cleavage, we performed a systematic analysis of the maturation of endogenous pri-miRNAs and their variants both in vitro and in vivo. We show that in addition to previously known features, the overall structural flexibility of pri-miRNA impact Drosha cleavage fidelity. Internal loops and nearby G · U wobble pairs on the pri-miRNA stem induce the use of non-canonical cleavage sites by Drosha, resulting in 5’ isomiR production. By analysing patient data deposited in the Cancer Genome Atlas, we provide evidence that alternative Drosha cleavage of pri-miRNAs is a tunable process that responds to the level of pri-miRNA-associated RNA-binding proteins. Together, our findings reveal that Drosha cleavage fidelity can be modulated by altering pri-miRNA structure, a potential mechanism underlying 5’ isomiR biogenesis in tumours. (Figure presented.).

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Forskning Sat, 01 Jan 2022 09:07:34 +0100 e32b92ec-c523-4454-807a-e1066ee7ade7
<![CDATA[Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=2ba08860-df0c-4fcb-b2af-474d16763a6b&tx_pure_pure5%5BshowType%5D=pub&cHash=31812ab68a54bf13a63f494f0ffb1692 Bofill-De Ros, X., Villanueva, E., Fillat, C. Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3'UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3'UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses.

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Forskning Thu, 01 Jan 2015 09:07:34 +0100 2ba08860-df0c-4fcb-b2af-474d16763a6b
<![CDATA[RNA targeting using CasRx can prevent aminoglycoside-induced hearing loss]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=87ad6f1e-1aca-4728-8666-6d6d79353a53&tx_pure_pure5%5BshowType%5D=pub&cHash=e5be1bb255bca3759102ff50bcb86bec Bofill-De Ros, X. Forskning Tue, 14 Jun 2022 09:07:34 +0200 87ad6f1e-1aca-4728-8666-6d6d79353a53 <![CDATA[AGO-bound mature miRNAs are oligouridylated by TUTs and subsequently degraded by DIS3L2]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=1f98064e-bbe6-434d-9eda-b3ef427c0920&tx_pure_pure5%5BshowType%5D=pub&cHash=37e2b62d9cf8a4d73b0d6dfa04f871c2 Yang, A., Shao, T. J., Bofill-De Ros, X., et al. MicroRNAs (miRNAs) associated with Argonaute proteins (AGOs) regulate gene expression in mammals. miRNA 3’ ends are subject to frequent sequence modifications, which have been proposed to affect miRNA stability. However, the underlying mechanism is not well understood. Here, by genetic and biochemical studies as well as deep sequencing analyses, we find that AGO mutations disrupting miRNA 3’ binding are sufficient to trigger extensive miRNA 3’ modifications in HEK293T cells and in cancer patients. Comparing these modifications in TUT4, TUT7 and DIS3L2 knockout cells, we find that TUT7 is more robust than TUT4 in oligouridylating mature miRNAs, which in turn leads to their degradation by the DIS3L2 exonuclease. Our findings indicate a decay machinery removing AGO-associated miRNAs with an exposed 3’ end. A set of endogenous miRNAs including miR-7, miR-222 and miR-769 are targeted by this machinery presumably due to target-directed miRNA degradation.

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Forskning Tue, 01 Dec 2020 09:07:34 +0100 1f98064e-bbe6-434d-9eda-b3ef427c0920
<![CDATA[Guidelines for the optimal design of miRNA-based shRNAs]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=59bba8bb-9707-4cc8-b112-b423fbf7a5b7&tx_pure_pure5%5BshowType%5D=pub&cHash=b7a6e40ffc7264b14a0e1593f06a5805 Bofill-De Ros, X., Gu, S. RNA interference (RNAi) is an extremely useful tool for inhibiting gene expression. It can be triggered by transfected synthetic small interfering RNA (siRNA) or by expressed small hairpin RNA (shRNA). The cellular machinery processes the latter into siRNA in vivo. shRNA is preferred or required in genetic screens and specific RNAi approaches in gene therapy settings. Despite its many successes, the field of shRNAs faces many challenges. Insufficient knockdowns and off-target effects become obstacles for shRNA usage in many applications. Numerous failures are triggered by pitfalls in shRNA design that is often associated with impoverished biogenesis. Here, based on current understanding of the miRNA maturation pathway, we discuss the principles of different shRNA design (pre-miRNA-like, pri-miRNA-like and Ago-shRNA) with an emphasis on the RNA structure. We also provide detailed instructions for an optimal design of pre-miRNA-like shRNA.

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Forskning Fri, 01 Jul 2016 09:07:34 +0200 59bba8bb-9707-4cc8-b112-b423fbf7a5b7
<![CDATA[Tumor IsomiR Encyclopedia (TIE)]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=246cb488-d318-4e81-812f-99245632ffe1&tx_pure_pure5%5BshowType%5D=pub&cHash=853f0d8256d0fce0542aab57ffdfcdcb Bofill-De Ros, X., Luke, B., Guthridge, R., Mudunuri, U., Loss, M., Gu, S. MicroRNAs (miRNAs) are master regulators of gene expression in cancers. Their sequence variants or isoforms (isomiRs) are highly abundant and possess unique functions. Given their short sequence length and high heterogeneity, mapping isomiRs can be challenging; without adequate depth and data aggregation, low frequency events are often disregarded. To address these challenges, we present the Tumor IsomiR Encyclopedia (TIE): a dynamic database of isomiRs from over 10 000 adult and pediatric tumor samples in The Cancer Genome Atlas (TCGA) and The Therapeutically Applicable Research to Generate Effective Treatments (TARGET) projects. A key novelty of TIE is its ability to annotate heterogeneous isomiR sequences and aggregate the variants obtained across all datasets. Results can be browsed online or downloaded as spreadsheets. Here, we show analysis of isomiRs of miR-21 and miR-30a to demonstrate the utility of TIE.

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Forskning Wed, 15 Sep 2021 09:07:34 +0200 246cb488-d318-4e81-812f-99245632ffe1
<![CDATA[Restoration of microRNA metabolism trigger robust antitumor responses]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=531e8b3e-ce00-40d9-a6bb-283a905bca0a&tx_pure_pure5%5BshowType%5D=pub&cHash=0774610c2e86ffb4474b8e7acd4d8ab2 Bofill-De Ros, X. Forskning Tue, 13 Sep 2022 09:07:34 +0200 531e8b3e-ce00-40d9-a6bb-283a905bca0a <![CDATA[Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=1ddea5d5-10fd-4f46-9d09-6e4c4e230049&tx_pure_pure5%5BshowType%5D=pub&cHash=6b58031e7d75f0a780068aa4115a75b5 Zhang, X., Malle, M. G., Thomsen, R. P., et al. DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

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Forskning Wed, 17 Apr 2024 09:07:34 +0200 1ddea5d5-10fd-4f46-9d09-6e4c4e230049
<![CDATA[3′ Uridylation Confers miRNAs with Non-canonical Target Repertoires]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=16343e10-5aad-4498-9f0e-e7adc5f9f2f8&tx_pure_pure5%5BshowType%5D=pub&cHash=6af17e9c7f91db2eb32f76ada7671e37 Yang, A., Bofill-De Ros, X., Shao, T. J., et al. Many microRNAs (miRNAs) exist alongside abundant miRNA isoforms (isomiRs), most of which arise from post-maturation sequence modifications such as 3′ uridylation. However, the ways in which these sequence modifications affect miRNA function remain poorly understood. Here, using human miR-27a in cell lines as a model, we discovered that a nonfunctional target site unable to base-pair extensively with the miRNA seed sequence can regain function when an upstream adenosine is able to base-pair with a post-transcriptionally added uridine in the miR-27a tail. This tail-U-mediated repression (TUMR) is abolished in cells lacking the uridylation enzymes TUT4 and TUT7, indicating that uridylation alters miRNA function by modulating target recognition. We identified a set of non-canonical targets in human cells that are specifically regulated by uridylated miR-27a. We provide evidence that TUMR expands the targets of other endogenous miRNAs. Our study reveals a function of uridylated isomiRs in regulating non-canonical miRNA targets. Yang et al. demonstrate that mRNAs lacking a seed pairing are repressed by miR-27a because of base-pairing between an upstream adenosine in the target and a non-templated U tail in miR-27a. They identify a large class of non-canonical targets regulated by uridylated miRNAs and reveal a novel function of 3′ isomiRs.

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Forskning Thu, 08 Aug 2019 09:07:34 +0200 16343e10-5aad-4498-9f0e-e7adc5f9f2f8
<![CDATA[Stress-induced microrna-708 impairs b-cell function and growth]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c7ab5959-eee3-4e58-8fbf-8e4da62ed6f3&tx_pure_pure5%5BshowType%5D=pub&cHash=16659d4418263982738385f06ee821dc Rodríguez-Comas, J., Moreno-Asso, A., Moreno-Vedia, J., et al. The pancreatic β-cell transcriptome is highly sensitive to external signals such as glucose oscillations and stress cues. MicroRNAs (miRNAs) have emerged as key factors in gene expression regulation. Here, we aimed to identify miRNAs that are modulated by glucose in mouse pancreatic islets.We identified miR-708 as the most upregulatedmiRNA in islets cultured at low glucose concentrations, a setting that triggers a strong stress response.miR-708 was also potently upregulated by triggering endoplasmic reticulum (ER) stress with thapsigargin and in islets of ob/ob mice. Low-glucose induction of miR-708 was blocked by treatment with the chemical chaperone 4-phenylbutyrate, uncovering the involvement of ER stress in this response. An integrative analysis identified neuronatin (Nnat) as a potential glucoseregulated target of miR-708. Indeed, Nnat expression was inversely correlated with miR-708 in islets cultured at different glucose concentrations and in ob/ob mouse islets and was reduced after miR-708 overexpression. Consistent with the role of Nnat in the secretory function of β-cells, miR-708 overexpression impaired glucosestimulated insulin secretion (GSIS), which was recovered by NNAT overexpression. Moreover, miR-708 inhibition recovered GSIS in islets cultured at low glucose. Finally, miR-708 overexpression suppressed β-cell proliferation and induced β-cell apoptosis. Collectively, our results provide a novel mechanism of glucose regulation of β-cell function and growth by repressing stress-induced miR-708.

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Forskning Fri, 01 Dec 2017 09:07:34 +0100 c7ab5959-eee3-4e58-8fbf-8e4da62ed6f3
<![CDATA[Structural Differences between Pri-miRNA Paralogs Promote Alternative Drosha Cleavage and Expand Target Repertoires]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=4ee11a5b-a161-4f35-a106-61699ee4bc8c&tx_pure_pure5%5BshowType%5D=pub&cHash=be1eeefb904cf71ff652d29710f3f16e Bofill-De Ros, X., Kasprzak, W. K., Bhandari, Y., et al. By studying the processing of pri-miR-9 family, Bofill-De Ros et al. demonstrate that the tertiary structure of pri-miRNA triggers alternative biogenesis. Drosha cleaves pri-miR-9-1 at an additional site because of its distorted and flexible lower stem, generating a 5′ isomiR that regulates a distinct set of genes in low-grade glioma.

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Forskning Tue, 08 Jan 2019 09:07:34 +0100 4ee11a5b-a161-4f35-a106-61699ee4bc8c
<![CDATA[TENT2, TUT4, and TUT7 selectively regulate miRNA sequence and abundance]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=65be5375-61de-4e2b-9bde-9bc7b3f91b4f&tx_pure_pure5%5BshowType%5D=pub&cHash=5d7fb2ee2b945c34a1c5d6d9e2f73ea7 Yang, A., Bofill-De Ros, X., Stanton, R., Shao, T. J., Villanueva, P., Gu, S. TENTs generate miRNA isoforms by 3’ tailing. However, little is known about how tailing regulates miRNA function. Here, we generate isogenic HEK293T cell lines in which TENT2, TUT4 and TUT7 are knocked out individually or in combination. Together with rescue experiments, we characterize TENT-specific effects by deep sequencing, Northern blot and in vitro assays. We find that 3’ tailing is not random but highly specific. In addition to its known adenylation, TENT2 contributes to guanylation and uridylation on mature miRNAs. TUT4 uridylates most miRNAs whereas TUT7 is dispensable. Removing adenylation has a marginal impact on miRNA levels. By contrast, abolishing uridylation leads to dysregulation of a set of miRNAs. Besides let-7, miR-181b and miR-222 are negatively regulated by TUT4/7 via distinct mechanisms while the miR-888 cluster is upregulated specifically by TUT7. Our results uncover the selective actions of TENTs in generating 3’ isomiRs and pave the way to investigate their functions.

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Forskning Thu, 01 Dec 2022 09:07:34 +0100 65be5375-61de-4e2b-9bde-9bc7b3f91b4f
<![CDATA[Novel, abundant Drosha isoforms are deficient in miRNA processing in cancer cells]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=93bc37a7-2d69-4979-aa18-aa491f671ecd&tx_pure_pure5%5BshowType%5D=pub&cHash=f303b30b6c8250385affc50427c50fc3 Dai, L., Hallmark, L., Bofill De Ros, X., et al. MicroRNAs (miRNAs) are a class of small noncoding RNAs about 22-nucleotide (nt) in length that collectively regulate more than 60% of coding genes. Aberrant miRNA expression is associated with numerous diseases, including cancer. miRNA biogenesis is licenced by the ribonuclease (RNase) III enzyme Drosha, the regulation of which is critical in determining miRNA levels. We and others have previously revealed that alternative splicing regulates the subcellular localization of Drosha. To further investigate the alternative splicing landscape of Drosha transcripts, we performed PacBio sequencing in different human cell lines. We identified two novel isoforms resulting from partial intron-retention in the region encoding the Drosha catalytic domain. One isoform (AS27a) generates a truncated protein that is unstable in cells. The other (AS32a) produces a full-length Drosha with a 14 amino acid insertion in the RIIID domain. By taking advantage of Drosha knockout cells in combination with a previously established reporter assay, we demonstrated that Drosha-AS32a lacks cleavage activity. Furthermore, neither Drosha-27a nor Drosha-32a were able to rescue miRNA expression in the Drosha knockout cells. Interestingly, both isoforms were abundantly detected in a wide range of cancer cell lines (up to 15% of all Drosha isoforms). Analysis of the RNA-seq data from over 1000 breast cancer patient samples revealed that the AS32a is relatively more abundant in tumours than in normal tissue, suggesting that AS32a may play a role in cancer development.

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Forskning Sun, 01 Nov 2020 09:07:34 +0100 93bc37a7-2d69-4979-aa18-aa491f671ecd
<![CDATA[QuagmiR]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=80937787-4612-446d-a8fe-3673da5a03ee&tx_pure_pure5%5BshowType%5D=pub&cHash=a92cca51d5ef0064405a82a2be69e9a5 Bofill-De Ros, X., Chen, K., Chen, S., et al. MicroRNAs (miRNAs) function as master regulators of gene expression. Recent studies demonstrate that miRNA isoforms (isomiRs) play a unique role in cancer development. Here, we present QuagmiR, the first cloud-based tool to analyze isomiRs from next generation sequencing data. Using a novel and flexible searching algorithm designed for the detection and annotation of heterogeneous isomiRs, it permits extensive customization of the query process and reference databases to meet the user 's diverse research needs.

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Forskning Wed, 01 May 2019 09:07:34 +0200 80937787-4612-446d-a8fe-3673da5a03ee
<![CDATA[Specifications of the ACMG/AMP Variant Classification Guidelines for Germline DICER1 Variant Curation]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=c7c17e83-d57c-4894-a118-bb711c8d5456&tx_pure_pure5%5BshowType%5D=pub&cHash=dbbfc05533e840d748302761e7f83ad9 Hatton, J. N., Frone, M. N., Cox, H. C., et al. Germline pathogenic variants in DICER1 predispose individuals to develop a variety of benign and malignant tumors. Accurate variant curation and classification are essential for reliable diagnosis of DICER1-related tumor predisposition and the identification of individuals who may benefit from surveillance. Since 2015, most labs have followed the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) sequence variant classification guidelines for DICER1 germline variant curation. However, these general guidelines lack gene-specific nuances and leave room for subjectivity. Consequently, a group of DICER1 experts joined ClinGen to form the DICER1 and miRNA-Processing Genes Variant Curation Expert Panel (VCEP) to create DICER1-specific ACMG/AMP guidelines for germline variant curation. The VCEP followed the FDA-approved ClinGen protocol for adapting and piloting these guidelines. A diverse set of 40 DICER1 variants were selected for piloting, including 14 known pathogenic/likely pathogenic (P/LP) variants, 12 known benign/likely benign (B/LB) variants, and 14 variants classified as variants of uncertain significance (VUS) or with conflicting interpretations in ClinVar. Clinically meaningful classifications (i.e., P, LP, LB, or B) were achieved for 82.5% (33/40) of the pilot variants, with 100% concordance among the known P/LP and known B/LB variants. Half of the VUS or conflicting variants were resolved with four variants classified as LB and three as LP. These results demonstrate that the DICER1-specific guidelines for germline variant curation effectively classify known pathogenic and benign variants while reducing the frequency of uncertain classifications. Individuals and labs curating DICER1 variants should consider adopting this classification framework to encourage consistency and improve objectivity.

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Forskning Sun, 01 Jan 2023 09:07:34 +0100 c7c17e83-d57c-4894-a118-bb711c8d5456
<![CDATA[Recent progress in miRNA biogenesis and decay]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=af03c8dd-164a-4776-88ea-ab9d1e909270&tx_pure_pure5%5BshowType%5D=pub&cHash=613449c6d052cfe05ad177fd4c5e6742 Bofill-De Ros, X., Vang Ørom, U. A. MicroRNAs are a class of small regulatory RNAs that mediate regulation of protein synthesis by recognizing sequence elements in mRNAs. MicroRNAs are processed through a series of steps starting from transcription and primary processing in the nucleus to precursor processing and mature function in the cytoplasm. It is also in the cytoplasm where levels of mature microRNAs can be modulated through decay mechanisms. Here, we review the recent progress in the lifetime of a microRNA at all steps required for maintaining their homoeostasis. The increasing knowledge about microRNA regulation upholds great promise as therapeutic targets.

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Forskning Mon, 01 Jan 2024 09:07:34 +0100 af03c8dd-164a-4776-88ea-ab9d1e909270
<![CDATA[Olfactory Epithelium Stimulation Using Rhythmic Nasal Air-Puffs Improves the Cognitive Performance of Individuals with Acute Sleep Deprivation]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=08b32546-7062-4145-8fe2-7cdec81a27d0&tx_pure_pure5%5BshowType%5D=pub&cHash=0afdf2cb7fcc239566ca6b7cd4646cc2 Riazi, H., Nazari, M., Raoufy, M. R., Mirnajafi-Zadeh, J., Shojaei, A. This study aimed to investigate the effects of intranasal air-puffing on cognitive impairments and brain cortical activity following one night of partial sleep deprivation (PSD) in adults. A total of 26 healthy adults underwent the numerical Stroop test (NST) and electroencephalography (EEG) before and after one night of PSD. Following PSD, subjects in the treatment group (n = 13) received nasal air-puffs (5 Hz, 3 min) before beginning the NST and EEG recording. Administration of nasal air-puffs in the treatment group restored the PSD-induced increase in error rate and decrease in reaction time and missing rate in the NST. Intranasal air-puffs recovered the PSD-induced augmentation of delta and theta power and the reduction of beta and gamma power in the EEG, particularly in the frontal lobes. Intranasal air-puffing also almost reversed the PSD-induced decrease in EEG signal complexity. Furthermore, it had a restorative effect on PSD-induced alteration in intra-default mode network functional connectivity in the beta and gamma frequency bands. Rhythmic nasal air-puffing can mitigate acute PSD-induced impairments in cognitive functions. It exerts part of its ameliorating effect by restoring neuronal activity in cortical brain areas involved in cognitive processing.

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Forskning Mon, 01 Apr 2024 09:07:34 +0200 08b32546-7062-4145-8fe2-7cdec81a27d0
<![CDATA[Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation]]> https://mbg.au.dk/forskning/publikationer?tx_pure_pure5%5Baction%5D=single&tx_pure_pure5%5Bcontroller%5D=Publications&tx_pure_pure5%5Bid%5D=4887be6e-a4a8-437d-b01b-16ddb90b44af&tx_pure_pure5%5BshowType%5D=pub&cHash=88269dc6b15d5bf71d4f7ad6e0730d6a Frain, K. M., Dedic, E., Nel, L., et al. The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

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Forskning Mon, 01 Apr 2024 09:07:34 +0200 4887be6e-a4a8-437d-b01b-16ddb90b44af