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  • 1.
    Abreu-Vieira, Gustavo
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fischer, Alexander W.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Hamburg, Germany.
    Mattsson, Charlotte
    de Jong, Jasper M. A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Shabalina, Irina G.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ryden, Mikael
    Laurencikiene, Jurga
    Arner, Peter
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cidea improves the metabolic profile through expansion of adipose tissue2015In: Nature Communications, E-ISSN 2041-1723, Vol. 6, article id 7433Article in journal (Refereed)
    Abstract [en]

    In humans, Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A) is highly but variably expressed in white fat, and expression correlates with metabolic health. Here we generate transgenic mice expressing human Cidea in adipose tissues (aP2-hCidea mice) and show that Cidea is mechanistically associated with a robust increase in adipose tissue expandability. Under humanized conditions (thermoneutrality, mature age and prolonged exposure to high-fat diet), aP2-hCidea mice develop a much more pronounced obesity than their wild-type littermates. Remarkably, the malfunctioning of visceral fat normally caused by massive obesity is fully overcome-perilipin 1 and Akt expression are preserved, tissue degradation is prevented, macrophage accumulation is decreased and adiponectin expression remains high. Importantly, the aP2-hCidea mice display enhanced insulin sensitivity. Our data establish a functional role for Cidea and suggest that, in humans, the association between Cidea levels in white fat and metabolic health is not only correlative but also causative.

  • 2. Acevedo, Nathalie
    et al.
    Benfeitas, Rui
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Katayama, Shintaro
    Bruhn, Sören
    Andersson, Anna
    Wikberg, Gustav
    Lundeberg, Lena
    Lindvall, Jessica M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Greco, Dario
    Kere, Juha
    Söderhäll, Cilla
    Scheynius, Annika
    Epigenetic alterations in skin homing CD4(+)CLA(+) T cells of atopic dermatitis patients2020In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 18020Article in journal (Refereed)
    Abstract [en]

    T cells expressing the cutaneous lymphocyte antigen (CLA) mediate pathogenic inflammation in atopic dermatitis (AD). The molecular alterations contributing to their dysregulation remain unclear. With the aim to elucidate putative altered pathways in AD we profiled DNA methylation levels and miRNA expression in sorted T cell populations -(CD4(+), -CD4(+)CD45RA(+) naive, -CD4(+)CLA(+), and -CD8(+)) from adult AD patients and healthy controls (HC). Skin homing -CD4(+)CLA(+) T cells from AD patients showed significant differences in DNA methylation in 40 genes compared to HC (p < 0.05). Reduced DNA methylation levels in the upstream region of the interleukin-13 gene (IL13) in -CD4(+)CLA(+) T cells from AD patients correlated with increased IL13 mRNA expression in these cells. Sixteen miRNAs showed differential expression in -CD4(+)CLA(+) T cells from AD patients targeting genes in 202 biological processes (p < 0.05). An integrated network analysis of miRNAs and CpG sites identified two communities of strongly interconnected regulatory elements with strong antagonistic behaviours that recapitulated the differences between AD patients and HC. Functional analysis of the genes linked to these communities revealed their association with key cytokine signaling pathways, MAP kinase signaling and protein ubiquitination. Our findings support that epigenetic mechanisms play a role in the pathogenesis of AD by affecting inflammatory signaling molecules in skin homing -CD4(+)CLA(+) T cells and uncover putative molecules participating in AD pathways.

  • 3. Acheva, Anna
    et al.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Caen Normandy, France.
    Sollazzo, Alice
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Launonen, Virpi
    Kamarainen, Meerit
    Presence of Stromal Cells Enhances Epithelial-to-Mesenchymal Transition (EMT) Induction in Lung Bronchial Epithelium after Protracted Exposure to Oxidative Stress of Gamma Radiation2019In: Oxidative Medicine and Cellular Longevity, ISSN 1942-0900, E-ISSN 1942-0994, Vol. 2019, article id 4120379Article in journal (Refereed)
    Abstract [en]

    The aim of the study was to investigate the role of a microenvironment in the induction of epithelial-to-mesenchymal transition (EMT) as a sign of early stages of carcinogenesis in human lung epithelial cell lines after protracted low-dose rate gamma-radiation exposures. BEAS-2B and HBEC-3KT lung cell lines were irradiated with low-dose rate gamma-rays (Cs-137, 1.4 or 14 mGy/h) to 0.1 or 1 Gy with or without adding TGF-beta. TGF-beta-treated samples were applied as positive EMT controls and tested in parallel to find out if the radiation has a potentiating effect on the EMT induction. To evaluate the effect of the stromal component, the epithelial cells were irradiated in cocultures with stromal MRC-9 lung fibroblasts. On day 3 post treatment, the EMT markers: alpha-SMA, vimentin, fibronectin, and E-cadherin, were analyzed. The oxidative stress levels were evaluated by 8-oxo-dG analysis in both epithelial and fibroblast cells. The protracted exposure to low Linear Energy Transfer (LET) radiation at the total absorbed dose of 1 Gy was able to induce changes suggestive of EMT. The results show that the presence of the stromal component and its signaling (TGF-beta) in the cocultures enhances the EMT. Radiation had a minor cumulative effect on the TGF-beta-induced EMT with both doses. The oxidative stress levels were higher than the background in both epithelial and stromal cells post chronic irradiation (0.1 and 1 Gy); as for the BEAS-2B cell line, the increase was statistically significant. We suggest that the induction of EMT in bronchial epithelial cells by radiation requires more than single acute exposure and the presence of stromal component might enhance the effect through free radical production and accumulation.

  • 4. Adori, Monika
    et al.
    Bhat, Sadam
    Gramignoli, Roberto
    Valladolid-Acebes, Ismael
    Bengtsson, Tore
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Uhlèn, Mathias
    Adori, Csaba
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Hepatic Innervations and Nonalcoholic Fatty Liver Disease2023In: Seminars in liver disease (Print), ISSN 0272-8087, E-ISSN 1098-8971, Vol. 43, no 02, p. 149-162Article, review/survey (Refereed)
    Abstract [en]

    Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder. Increased sympathetic (noradrenergic) nerve tone has a complex role in the etiopathomechanism of NAFLD, affecting the development/progression of steatosis, inflammation, fibrosis, and liver hemodynamical alterations. Also, lipid sensing by vagal afferent fibers is an important player in the development of hepatic steatosis. Moreover, disorganization and progressive degeneration of liver sympathetic nerves were recently described in human and experimental NAFLD. These structural alterations likely come along with impaired liver sympathetic nerve functionality and lack of adequate hepatic noradrenergic signaling. Here, we first overview the anatomy and physiology of liver nerves. Then, we discuss the nerve impairments in NAFLD and their pathophysiological consequences in hepatic metabolism, inflammation, fibrosis, and hemodynamics. We conclude that further studies considering the spatial-temporal dynamics of structural and functional changes in the hepatic nervous system may lead to more targeted pharmacotherapeutic advances in NAFLD.

  • 5.
    Akuwudike, Pamela
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    López-Riego, Milagrosa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dehours, Cloé
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Polytech Angers l École d’Ingénieurs, France.
    Lundholm, Lovisa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Impact of fractionated cisplatin and radiation treatment on cell growth and accumulation of DNA damage in two normal cell types differing in origin2023In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, article id 14891Article in journal (Refereed)
    Abstract [en]

    Evidence on the impact of chemotherapy on radiotherapy-induced second malignant neoplasms is controversial. We estimated how cisplatin modulates the in vitro response of two normal cell types to fractionated radiation. AHH-1 lymphoblasts and VH10 fibroblasts were irradiated at 1 Gy/fraction 5 and 3 times per week during 12 and 19 days, respectively, and simultaneously treated with 0.1, 0.2, 0.4, 0.8, 1.7 and 3.3 µM of cisplatin twice a week. Cell growth during treatment was monitored. Cell growth/cell death and endpoints related to accumulation of DNA damage and, thus, carcinogenesis, were studied up to 21 days post treatment in cells exposed to radiation and the lowest cisplatin doses. Radiation alone significantly reduced cell growth. The impact of cisplatin alone below 3.3 µM was minimal. Except the lowest dose of cisplatin in VH10 cells, cisplatin reduced the inhibitory effect of radiation on cell growth. Delayed cell death was highest in the combination groups while the accumulation of DNA damage did not reveal a clear pattern. In conclusion, fractionated, concomitant exposure to radiation and cisplatin reduces the inhibitory effect of radiation on cell proliferation of normal cells and does not potentiate delayed effects resulting from accumulation of DNA damage.

  • 6.
    Akuwudike, Pamela
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Tartas, Adrianna
    López-Riego, Milagrosa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Toma-Daşu, Iuliana
    Stockholm University, Faculty of Science, Department of Physics. Karolinska Institutet, Sweden.
    Wojcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Lundholm, Lovisa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cell Type-Specific Patterns in the Accumulation of DNA Damage Following Multifractional Radiation Exposure2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 21, article id 12861Article in journal (Refereed)
    Abstract [en]

    Predicting the risk of second malignant neoplasms is complicated by uncertainties regarding the shape of the dose–response relationship at high doses. Limited understanding of the competitive relationship between cell killing and the accumulation of DNA lesions at high doses, as well as the effects of other modulatory factors unique to radiation exposure during radiotherapy, such as dose heterogeneity across normal tissue and dose fractionation, contribute to these uncertainties. The aim of this study was to analyze the impact of fractionated irradiations on two cell systems, focusing on the endpoints relevant for cancer induction. To simulate the heterogeneous dose distribution across normal tissue during radiotherapy, exponentially growing VH10 fibroblasts and AHH-1 lymphoblasts were irradiated with 9 and 12 fractions (VH10) and 10 fractions (AHH-1) at 0.25, 0.5, 1, or 2 Gy per fraction. The effects on cell growth, cell survival, radiosensitivity and the accumulation of residual DNA damage lesions were analyzed as functions of dose per fraction and the total absorbed dose. Residual γH2AX foci and other DNA damage markers (micronuclei, nuclear buds, and giant nuclei) were accumulated at high doses in both cell types, but in a cell type-dependent manner. The competitive relationship between cell killing and the accumulation of carcinogenic DNA damage following multifractional radiation exposure is cell type-specific.

  • 7. Alberro-Brage, Andres
    et al.
    Kryvenko, Vitalii
    Malainou, Christina
    Guenther, Stefan
    Morty, Rory E.
    Seeger, Werner
    Herold, Susanne
    Samakovlis, Christos
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Vadasz, Istvan
    Influenza virus decreases albumin uptake and megalin expression in alveolar epithelial cells2023In: Frontiers in Immunology, E-ISSN 1664-3224, Vol. 14, article id 1260973Article in journal (Refereed)
    Abstract [en]

    Introduction

    Acute respiratory distress syndrome (ARDS) is a common complication of influenza virus (IV) infection. During ARDS, alveolar protein concentrations often reach 40-90% of plasma levels, causing severe impairment of gas exchange and promoting deleterious alveolar remodeling. Protein clearance from the alveolar space is at least in part facilitated by the multi-ligand receptor megalin through clathrin-mediated endocytosis.

    Methods

    To investigate whether IV infection impairs alveolar protein clearance, we examined albumin uptake and megalin expression in MLE-12 cells and alveolar epithelial cells (AEC) from murine precision-cut lung slices (PCLS) and in vivo, under IV infection conditions by flow cytometry and western blot. Transcriptional levels from AEC and broncho-alveolar lavage (BAL) cells were analyzed in an in-vivo mouse model by RNAseq.

    Results

    IV significantly downregulated albumin uptake, independently of activation of the TGF- β1/GSK3β axis that has been previously implicated in the regulation of megalin function. Decreased plasma membrane abundance, total protein levels, and mRNA expression of megalin were associated with this phenotype. In IV-infected mice, we identified a significant upregulation of matrix metalloproteinase (MMP)-14 in BAL fluid cells. Furthermore, the inhibition of this protease partially recovered total megalin levels and albumin uptake.

    Discussion

    Our results suggest that the previously described MMP-driven shedding mechanisms are potentially involved in downregulation of megalin cell surface abundance and clearance of excess alveolar protein. As lower alveolar edema protein concentrations are associated with better outcomes in respiratory failure, our findings highlight the therapeutic potential of a timely MMP inhibition in the treatment of IV-induced ARDS.

  • 8.
    Alkemar, Gunnar
    Stockholm University, Faculty of Science, Wenner-Gren Institute for Experimental Biology.
    Ribosome and ribosomal RNA Structure: An experimental and computational analysis of expansion segments in eukaryotic ribosomal RNA2008Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Ribosomes are large ribonucleoprotein complexes which incorporate amino acids into peptide chains during translational process in all types of living cells. The eukaryotic ribosome is larger compared to its prokaryotic counterpart. The size differences are due to a larger protein part and that the rRNA contains eukaryote specific expansion segments (ES). Cryo-EM reconstruction has visualized many ES on the ribosomal surface which have given clues about function and structural features. However, the secondary structures of most ES are unknown or ill defined. In this thesis, the secondary and also to a certain extent the tertiary structures of several ES are determined by using computational methods and biochemical experimental techniques. The juxtaposition of ES6 close to ES3 in the Cryo-EM image of the yeast ribosome suggested that ES3 and ES6 might interact. A computational analysis of more than 2900 sequences shows that a complementary helical region of seven to nine contiguous base pairs can form between ES3 and ES6 in almost all analyzed sequences. Biochemical in situ experiments support the proposed interaction. Secondary structure models are presented for ES3 and ES6 in 18S rRNA and ES39 in 28S rRNA, where homologous structural elements could be modeled in the experimentally analyzed ribosomes from fungi, plants and mammals. The structure models were further supported by computational methods where the ES6 structure and the ES39 structure could be formed in more than 6000 and 900 sequences respectively. A tertiary structure model of ES3 and ES6 including the helical interaction is presented. An in vitro transcribed and folded ES6 sequence differed from that observed in situ, suggesting that chaperones, ribosomal proteins, and/or the tertiary rRNA interaction could be involved in the in vivo folding of ES6. An analysis of the similarities between ES39 structures suggests that it might be under selective constraint to preserve its secondary structure.

  • 9.
    Alsheikhly, Abdul-Razzak
    Stockholm University.
    Virus mediated induction of antibody independent cytotoxicity (VDCC) and enhancement of antibody dependent cytotoxicity (ADCC) in human lymphocytes in vitro1984Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Addition in vitro of small amounts of paramyxoviruses (Mumps or Sendai) to lymphocytes from healthy human donors induces a strong and non-selective cytotoxicity against a variety of target cells (VDCC, virus dependent cellular cytotoxicity). VDCC-like reactions are believed to play a role in vivo in the defense against virus infection and/or the causation of the tissue lesions associated with virus disease. The aim of this study was to investigate the mechanism of VDCC induction, its relationship to other lymphocyte mediated cytotoxic reactions and the nature of the effector cells involved.

    UV-inactivated virions induce VDCC as efficiently as live virus, indicating that it is not dependent on infection of either lymphocytes or target cells. Removal from the virions of the HN-surface glycoprotein carrying hemagglutination and neuraminidase activities abrogated VDCC. Removal of the F protein (F=fusion) was less efficient. When the lymphocytes were treated with solubilized HN- or F proteins, the HNprotein had full capacity to induce VDCC while the F protein was inactive. The importance of the HN-protein for VDCC was also supported by inhibition experiments with virus specific antibodies. Monoclonal anti-HN antibodies (mumps) inhibited VDCC whereas anti-F, anti-M (membrane) or anti-NP (nucleoprotein) antibodies did not. By using a panel of monoclonals directed to several distinct determinants of the HN-polypeptide, it was shown that at least 3 serologically defined structures of this protein were involved in VDCC induction. These structures appeared to be different from those involved in hemagglutination, hemolysis, neuraminidase activity and infectivity of the virus.

    When assayed at the level of the effector cell population (51Cr-release), VDCC was reflected by increased target cell lysis. At the effector cell level (single cell conjugate assay), VDCC was the expression of recruitment of both target-binding cells and target killing cells. In short term assays, virus treatment of the lymphocytes did not increase their recycling capacity, confirming that VDCC was primarily due to recruitment. Virus treatment of lymphocytes induces the release of interferon, known to enhance their cytotoxicity. VDCC induction appears to be independent of interferon. However, an activating effect of interferon in later phases of VDCC is not excluded.

    Cell fractionation experiments and single cell assays in combination with surface marker studies showed that VDCC effector cells were heterogeneous, including both large granular lymphocytes (LGL) and small to medium sized lymphocytes with Tcell characteristics. The cytotoxic effector cells recruited by virus comprised both lymphocytes bearing the LGL-associated HNK-1 antigen and lymphocytes bearing T-cell associated antigens. In absolute numbers, the majority of die effector cells had T-cell characteristics. However, the proportion of the latter differed for target cells of different types, suggesting that the target cells play a role in effector cell selection.

    VDCC is non-selective and is not MHC restricted, indicating that it is not mediated by specifically cytotoxic T-lymphocytes (CTL). Experiments with cord blood lymphocytes also showed that VDCC was not induced through specific or polyclonal activation by some viral material. VDCC induction is also independent of the participation of anti-target cell antibodies. However, virus treatment of the lymphocytes strongly enhanced their antibody dependent cytotoxicity (ADCC) both against VDCC susceptible tumor target cells and against VDCC resistant erythrocytic target cells. ADCC enhancement was a reflection of effector cell recruitment, primarily involving lymphocytes bearing T-cell markers. It was inhibited by monoclonal antibodies to the HN-protein. Taken together, these experiments showed that virus enhances ADCC by improving the effector cell target cell contacts necessary for cytotoxicity expression. This also results in a significant reduction in the amounts of antitarget cell antibodies required for the triggering of cytotoxicity.

  • 10. Asami, Jinta
    et al.
    Kimura, Kanako Terakado
    Fujita-Fujiharu, Yoko
    Ishida, Hanako
    Zhang, Zhikuan
    Nomura, Yayoi
    Liu, Kehong
    Uemura, Tomoko
    Sato, Yumi
    Ono, Masatsugu
    Yamamoto, Masaki
    Noda, Takeshi
    Shigematsu, Hideki
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Iwata, So
    Shimizu, Toshiyuki
    Nomura, Norimichi
    Ohto, Umeharu
    Structure of the bile acid transporter and HBV receptor NTCP2022In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 606, no 7916, p. 1021-1026Article in journal (Refereed)
    Abstract [en]

    Chronic infection with hepatitis B virus (HBV) affects more than 290 million people worldwide, is a major cause of cirrhosis and hepatocellular carcinoma, and results in an estimated 820,000 deaths annually. For HBV infection to be established, a molecular interaction is required between the large glycoproteins of the virus envelope (known as LHBs) and the host entry receptor sodium taurocholate co-transporting polypeptide (NTCP), a sodium-dependent bile acid transporter from the blood to hepatocytes. However, the molecular basis for the virus–transporter interaction is poorly understood. Here we report the cryo-electron microscopy structures of human, bovine and rat NTCPs in the apo state, which reveal the presence of a tunnel across the membrane and a possible transport route for the substrate. Moreover, the cryo-electron microscopy structure of human NTCP in the presence of the myristoylated preS1 domain of LHBs, together with mutation and transport assays, suggest a binding mode in which preS1 and the substrate compete for the extracellular opening of the tunnel in NTCP. Our preS1 domain interaction analysis enables a mechanistic interpretation of naturally occurring HBV-insusceptible mutations in human NTCP. Together, our findings provide a structural framework for HBV recognition and a mechanistic understanding of sodium-dependent bile acid translocation by mammalian NTCPs. 

  • 11. Asp, Michaela
    et al.
    Giacomello, Stefania
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Larsson, Ludvig
    Wu, Chenglin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Fürth, Daniel
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wärdell, Eva
    Custodio, Joaquin
    Reimegård, Johan
    Salmén, Fredrik
    Österholm, Cecilia
    Ståhl, Patrik L.
    Sundström, Erik
    Åkesson, Elisabet
    Bergmann, Olaf
    Bienko, Magda
    Månsson-Broberg, Agneta
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sylvén, Christer
    Lundeberg, Joakim
    A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart2019In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 179, no 7, p. 1647-1660Article in journal (Refereed)
    Abstract [en]

    The process of cardiac morphogenesis in humans is incompletely understood. Its full characterization requires a deep exploration of the organ-wide orchestration of gene expression with a single-cell spatial resolution. Here, we present a molecular approach that reveals the comprehensive transcriptional landscape of cell types populating the embryonic heart at three developmental stages and that maps cell-type-specific gene expression to specific anatomical domains. Spatial transcriptomics identified unique gene profiles that correspond to distinct anatomical regions in each developmental stage. Human embryonic cardiac cell types identified by single-cell RNA sequencing confirmed and enriched the spatial annotation of embryonic cardiac gene expression. In situ sequencing was then used to refine these results and create a spatial subcellular map for the three developmental phases. Finally, we generated a publicly available web resource of the human developing heart to facilitate future studies on human cardiogenesis.

  • 12. Assadi, Ghazaleh
    et al.
    Vesterlund, Liselotte
    Bonfiglio, Ferdinando
    Mazzurana, Luca
    Cordeddu, Lina
    Schepis, Danika
    Mjösberg, Jenny
    Ruhrmann, Sabrina
    Fabbri, Alessia
    Vukojevic, Vladana
    Percipalle, Piergiorgio
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. New York University Abu Dhabi, United Arab Emirates.
    Salomons, Florian A.
    Laurencikiene, Jurga
    Törkvist, Leif
    Halfvarson, Jonas
    D'Amato, Mauro
    Functional Analyses of the Crohn's Disease Risk Gene LACC12016In: PLOS ONE, E-ISSN 1932-6203, Vol. 11, no 12, article id e0168276Article in journal (Refereed)
    Abstract [en]

    Background Genetic variation in the Laccase (multicopper oxidoreductase) domain-containing 1 (LACC1) gene has been shown to affect the risk of Crohn's disease, leprosy and, more recently, ulcerative colitis and juvenile idiopathic arthritis. LACC1 function appears to promote fatty-acid oxidation, with concomitant inflammasome activation, reactive oxygen species production, and anti-bacterial responses in macrophages. We sought to contribute to elucidating LACC1 biological function by extensive characterization of its expression in human tissues and cells, and through preliminary analyses of the regulatory mechanisms driving such expression. Methods We implemented Western blot, quantitative real-time PCR, immunofluorescence microscopy, and flow cytometry analyses to investigate fatty acid metabolism-immune nexus (FAMIN; the LACC1 encoded protein) expression in subcellular compartments, cell lines and relevant human tissues. Gene-set enrichment analyses were performed to initially investigate modulatory mechanisms of LACC1 expression. A small-interference RNA knockdown in vitro model system was used to study the effect of FAMIN depletion on peroxisome function. Results FAMIN expression was detected in macrophage-differentiated THP-1 cells and several human tissues, being highest in neutrophils, monocytes/macrophages, myeloid and plasmacytoid dendritic cells among peripheral blood cells. Subcellular co-localization was exclusively confined to peroxisomes, with some additional positivity for organelle endomembrane structures. LACC1 co-expression signatures were enriched for genes involved in peroxisome proliferator-activated receptors (PPAR) signaling pathways, and PPAR ligands downregulated FAMIN expression in in vitro model systems. Conclusion FAMIN is a peroxisome-associated protein with primary role(s) in macrophages and other immune cells, where its metabolic functions may be modulated by PPAR signaling events. However, the precise molecular mechanisms through which FAMIN exerts its biological effects in immune cells remain to be elucidated.

  • 13.
    Attoff, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Johansson, Ylva
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Cediel-Ulloa, Andrea
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Karolinska Institutet, Sweden.
    Gupta, Rajinder
    Caiment, Florian
    Gliga, Anda
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Uppsala University, Sweden.
    Acrylamide alters CREB and retinoic acid signalling pathways during differentiation of the human neuroblastoma SH-SY5Y cell line2020In: Scientific Reports, E-ISSN 2045-2322, Vol. 10, no 1, article id 16714Article in journal (Refereed)
    Abstract [en]

    Acrylamide (ACR) is a known neurotoxicant which crosses the blood-brain barrier, passes the placenta and has been detected in breast milk. Hence, early-life exposure to ACR could lead to developmental neurotoxicity. The aim of this study was to elucidate if non-cytotoxic concentrations of ACR alter neuronal differentiation by studying gene expression of markers significant for neurodevelopment in the human neuroblastoma SH-SY5Y cell model. Firstly, by using RNASeq we identified two relevant pathways that are activated during 9 days of retinoic acid (RA) induced differentiation i.e. RA receptor (RAR) activation and the cAMP response element-binding protein (CREB) signalling pathways. Next, by qPCR we showed that 1 and 70 mu M ACR after 9 days exposure alter the expression of 13 out of 36 genes in the RAR activation pathway and 18 out of 47 in the CREB signalling pathway. Furthermore, the expression of established neuronal markers i.e. BDNF, STXBP2, STX3, TGFB1 and CHAT were down-regulated. Decreased protein expression of BDNF and altered ratio of phosphorylated CREB to total CREB were confirmed by western blot. Our results reveal that micromolar concentrations of ACR sustain proliferation, decrease neurite outgrowth and interfere with signalling pathways involved in neuronal differentiation in the SH-SY5Y cell model.

  • 14.
    Attoff, Kristina
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Kertika, Dimitra
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lundqvist, Jessica
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Oredsson, S.
    Forsby, Anna
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Acrylamide affects proliferation and differentiation of the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y2016In: Toxicology in Vitro, ISSN 0887-2333, E-ISSN 1879-3177, Vol. 35, p. 100-111Article in journal (Refereed)
    Abstract [en]

    Acrylamide is a well-known neurotoxic compound and people get exposed to the compound by food consumption and environmental pollutants. Since acrylamide crosses the placenta barrier, the fetus is also being exposed resulting in a risk for developmental neurotoxicity. In this study, the neural progenitor cell line C17.2 and the neuroblastoma cell line SH-SY5Y were used to study proliferation and differentiation as alerting indicators for developmental neurotoxicity. For both cell lines, acrylamide reduced the number of viable cells by reducing proliferation and inducing cell death in undifferentiated cells. Acrylamide concentrations starting at 10 fM attenuated the differentiation process in SH-SY5Y cells by sustaining cell proliferation and neurite outgrowth was reduced at concentrations from 10 pM. Acrylamide significantly reduced the number of neurons starting at 1 mu M and altered the ratio between the different phenotypes in differentiating C17.2 cell cultures. Ten micromolar of acrylamide also reduced the expression of the neuronal and astrocyte biomarkers. Although the neurotoxic concentrations in the femtomolar range seem to be specific for the SH-SY5Y cell line, the fact that micromolar concentrations of acrylamide seem to attenuate the differentiation process in both cell lines raises the interest to further investigations on the possible developmental neurotoxicity of acrylamide.

  • 15. Babini, Gabriele
    et al.
    Baiocco, Giorgio
    Barbieri, Sofia
    Morini, Jacopo
    Sangsuwan, Traimate
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Haghdoost, Siamak
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University of Caen Normandy, France.
    Yentrapalli, Ramesh
    Azimzadeh, Omid
    Rombouts, Charlotte
    Aerts, An
    Quintens, Roel
    Ebrahimian, Teni
    Benotmane, Mohammed Abderrafi
    Ramadan, Raghda
    Baatout, Sarah
    Tapio, Soile
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ottolenghi, Andrea
    A systems radiation biology approach to unravel the role of chronic low-dose-rate gamma-irradiation in inducing premature senescence in endothelial cells2022In: PLOS ONE, E-ISSN 1932-6203, Vol. 17, no 3, article id e0265281Article in journal (Refereed)
    Abstract [en]

    Purpose

    The aim of this study was to explore the effects of chronic low-dose-rate gamma-radiation at a multi-scale level. The specific objective was to obtain an overall view of the endothelial cell response, by integrating previously published data on different cellular endpoints and highlighting possible different mechanisms underpinning radiation-induced senescence.

    Materials and methods

    Different datasets were collected regarding experiments on human umbilical vein endothelial cells (HUVECs) which were chronically exposed to low dose rates (0, 1.4, 2.1 and 4.1 mGy/h) of gamma-rays until cell replication was arrested. Such exposed cells were analyzed for different complementary endpoints at distinct time points (up to several weeks), investigating cellular functions such as proliferation, senescence and angiogenic properties, as well as using transcriptomics and proteomics profiling. A mathematical model was proposed to describe proliferation and senescence.

    Results

    Simultaneous ceasing of cell proliferation and senescence onset as a function of time were well reproduced by the logistic growth curve, conveying shared equilibria between the two endpoints. The combination of all the different endpoints investigated highlighted a dose-dependence for prematurely induced senescence. However, the underpinning molecular mechanisms appeared to be dissimilar for the different dose rates, thus suggesting a more complex scenario.

    Conclusions

    This study was conducted integrating different datasets, focusing on their temporal dynamics, and using a systems biology approach. Results of our analysis highlight that different dose rates have different effects in inducing premature senescence, and that the total cumulative absorbed dose also plays an important role in accelerating endothelial cell senescence.

  • 16. Baniol, Marion
    et al.
    Murganti, Francesca
    Smialowska, Agata
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Panula, Joni
    Lázár, Enikő
    Brockman, Viveka
    Giatrellis, Sarantis
    Derks, Wouter
    Bergmann, Olaf
    Identification and characterization of distinct cell cycle stages in cardiomyocytes using the FUCCI transgenic system2021In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 408, no 2, article id 112880Article in journal (Refereed)
    Abstract [en]

    Understanding the regulatory mechanism by which cardiomyocyte proliferation transitions to endoreplication and cell cycle arrest during the neonatal period is crucial for identifying proproliferative factors and developing regenerative therapies. We used a transgenic mouse model based on the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system to isolate and characterize cycling cardiomyocytes at different cell cycle stages at a single-cell resolution. Single-cell transcriptome analysis of cycling and noncycling cardiomyocytes was performed at postnatal days 0 (P0) and 7 (P7). The FUCCI system proved to be efficient for the identification of cycling cardiomyocytes with the highest mitotic activity at birth, followed by a gradual decline in the number of cycling and mitotic cardiomyocytes during the neonatal period. Cardiomyocytes showed premature cell cycle exit at G1/S shortly after birth and delayed G1/S progression during endoreplication at P7. Single-cell RNA-seq confirmed previously described signaling pathways involved in cardiomyocyte proliferation (Erbb2 and Hippo/YAP), and maturation-related transcriptional changes during postnatal development, including the metabolic switch from glycolysis to fatty acid oxidation in cardiomyocytes. Importantly, we generated transcriptional profiles specific to cell division and endoreplication in cardiomyocytes at different developmental stages that may facilitate the identification of genes important for adult cardiomyocyte proliferation and heart regeneration. In conclusion, the FUCCI mouse provides a valuable system to study cardiomyocyte cell cycle activity at single cell resolution that can help to decipher the switch from cardiomyocyte proliferation to endoreplication, and to revert this process to facilitate endogenous repair.

  • 17. Barnekow, Elin
    et al.
    Hasslow, Johan
    Liu, Wen
    Bryant, Patrick
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). Karolinska Institutet, Sweden.
    Thutkawkorapin, Jessada
    Wendt, Camilla
    Czene, Kamila
    Hall, Per
    Margolin, Sara
    Lindblom, Annika
    A Swedish Familial Genome-Wide Haplotype Analysis Identified Five Novel Breast Cancer Susceptibility Loci on 9p24.3, 11q22.3, 15q11.2, 16q24.1 and Xq21.312023In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 5, article id 4468Article in journal (Refereed)
    Abstract [en]

    Most breast cancer heritability is unexplained. We hypothesized that analysis of unrelated familial cases in a GWAS context could enable the identification of novel susceptibility loci. In order to examine the association of a haplotype with breast cancer risk, we performed a genome-wide haplotype association study using a sliding window analysis of window sizes 1–25 SNPs in 650 familial invasive breast cancer cases and 5021 controls. We identified five novel risk loci on 9p24.3 (OR 3.4; p 4.9 × 10−11), 11q22.3 (OR 2.4; p 5.2 × 10−9), 15q11.2 (OR 3.6; p 2.3 × 10−8), 16q24.1 (OR 3; p 3 × 10−8) and Xq21.31 (OR 3.3; p 1.7 × 10−8) and confirmed three well-known loci on 10q25.13, 11q13.3, and 16q12.1. In total, 1593 significant risk haplotypes and 39 risk SNPs were distributed on the eight loci. In comparison with unselected breast cancer cases from a previous study, the OR was increased in the familial analysis in all eight loci. Analyzing familial cancer cases and controls enabled the identification of novel breast cancer susceptibility loci.

  • 18.
    Behm, Mikaela
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholms universitet.
    Regulation of RNA Editing: The impact of inosine on the neuronal transcriptome2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The transcriptome of the mammalian brain is extensively modified by adenosine to inosine (A-to-I) nucleotide conversion by two adenosine deaminases (ADAR1 and ADAR2). As adenosine and inosine have different base pairing properties, A-to-I RNA editing shapes the functional output of both coding and non-coding RNAs (ncRNAs) in the brain. The aim of this thesis was to identify editing events in small regulatory ncRNAs (miRNAs) and to determine their temporal and spatial editing status in the developing and adult mouse brain. To do this, we initially analyzed the editing status of miRNAs from different developmental time points of the mouse brain. We detected novel miRNA substrates subjected to A-to-I editing and found a general increase in miRNA editing during brain development, implicating a more stringent control of miRNAs as the brain matures. Most of the edited miRNAs were found to be transcribed as a single long consecutive transcript from a large gene cluster. However, maturation from this primary miRNA (pri-miRNA) transcript into functional forms of miRNAs is regulated individually, and might be influenced by the ADAR proteins in an editing independent matter. We also found that edited miRNAs were highly expressed at the synapse, implicating a role as local regulators of synaptic translation. We further show that the increase in editing during development is explained by a gradual accumulation of the ADAR enzymes in the nucleus. Specifically for ADAR2, we found a developmentally increasing interaction with two factors, importin-α4 and Pin1, that facilitate nuclear localization of the editing enzyme. We have also found that selectively edited stem loops often are flanked by other long stem loop structures that induce editing in cis. This may explain why multiple pri-miRNAs are edited within the same cluster. In conclusion, this thesis has significantly increased the understanding of the dynamics of both editing substrates and enzymes in the developing and mature brain.

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  • 19.
    Behm, Mikaela
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wahlstedt, Helene
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Widmark, Albin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Eriksson, Maria
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Öhman, Marie
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Accumulation of nuclear ADAR2 regulates A-to-I RNA editing during neuronal development2017In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 130, p. 745-753Article in journal (Refereed)
    Abstract [en]

    Adenosine to inosine (A-to-I) RNA editing is important for a functional brain, and most known sites that are subject to selective RNA editing have been found to result in diversified protein isoforms that are involved in neurotransmission. In the absence of the active editing enzymes ADAR1 or ADAR2 (also known as ADAR and ADARB1, respectively), mice fail to survive until adulthood. Nuclear A-to-I editing of neuronal transcripts is regulated during brain development, with low levels of editing in the embryo and a dramatic increase after birth. Yet, little is known about the mechanisms that regulate editing during development. Here, we demonstrate lower levels of ADAR2 in the nucleus of immature neurons than in mature neurons. We show that importin-a4 (encoded by Kpna3), which increases during neuronal maturation, interacts with ADAR2 and contributes to the editing efficiency by bringing it into the nucleus. Moreover, we detect an increased number of interactions between ADAR2 and the nuclear isomerase Pin1 as neurons mature, which contribute to ADAR2 protein stability. Together, these findings explain how the nuclear editing of substrates that are important for neuronal function can increase as the brain develops. 

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  • 20. Bell, Thomas J.
    et al.
    Eiríksdóttir, Emelía
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Eberwine, James
    PAIR technology: exon-specific RNA binding protein isolation in live cells2011In: Cell-penetrating peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2011, p. 473-486Chapter in book (Refereed)
    Abstract [en]

    RNA-binding proteins (RBPs) are fundamental regulatory proteins for all forms of transcriptional and posttranscriptional control of gene expression. However, isolating RBPs is technically challenging for investigators. Currently, the most widely used techniques to isolate RBPs are in vitro biochemical approaches. Although these approaches have been useful, they have several limitations. One key limitation to using in vitro biochemical approaches is that RBP–RNA interactions are isolated under nonbiological conditions. Here we review a novel experimental approach to identify RBPs called peptide nucleic acid (PNA)-assisted identification of RBPs (PAIR) technology (Zielinski et al., Proc Natl Acad Sci USA 103:1557–1562, 2006). This technology has two significant advantages over traditional approaches. (1) It overcomes the in vitro limitation of biochemical approaches by allowing investigators to isolate RBP–RNA interactions under in vivo conditions. (2) This technology is highly mRNA specific; it isolates RBPs in an exon-specific manner. By selectively targeting alternatively spliced exons with PAIR technology, investigators can isolate splice variant-specific and mRNA region-specific (5-UTR and 3-UTR) RBP complexes for any mRNA of interest.

  • 21.
    Bengtsson, Johanna
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The impact of cytochrome P4501-inhibitors on aryl hydrocarbon receptor signaling2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The aryl hydrocarbon receptor (AHR) best known as a ligand-activated transcription factor that mediates toxic responses to xenobiotics such as dioxins, is also activated by certain endogenous compounds. Activation of the AHR up-regulates transcription of a large number of genes, including those encoding members of the cytochrome P450 1 family of enzymes (CYP1s). Although the AHR has been shown to be involved in several normal processes, its physiological role remains elusive. The endogenous ligand 6-formylindolo[3,2-b]carbazole (FICZ), formed from tryptophan, is present in cell culture media and biological specimens. FICZ is an excellent substrate for CYP1 enzymes and together FICZ/AHR/CYP1A1 interactions constitute an auto regulatory feedback loop that controls AHR signaling. A vast number of compounds that inhibit CYP1 enzymes have been reported to be AHR activators, even though they have little or no affinity for the receptor. We hypothesized, that their agonistic effects are dependent on the presence of background levels of FICZ. To test this, AHR signaling in different cell systems exposed to FICZ and/or inhibitors was assessed by measuring EROD activity and CYP1A1 transcription. In addition to a commercial culture medium, a medium free of background levels of FICZ was used. Activation of AHR by of a diverse set of CYP1A1 inhibitors did require FICZ in the culture medium. Furthermore, the compounds tested both prolonged and potentiated FICZ-induced receptor signaling. On the basis of these observations we propose that a compound may activate AHR signaling indirectly by inhibiting CYP1A1 and thereby attenuating the metabolism of FICZ. This mechanism was confirmed for certain polyphenols and pharmaceuticals. Surprisingly, the activating capacity and potentiating effect of two pharmaceuticals on AHR signaling could not be explained by the mechanism proposed, and we speculated that in these cases the agonistic effect might involve interactions of the cellular antioxidant response with the basic transcription machinery. Together, our observations provide a mechanistic explanation as to how compounds that inhibit CYP1A1 can activate AHR signaling. They also indicate that the general perception of the binding pocket of AHR as promiscuous, is probably wrong. The fact that indirect activation of AHR may cause sustained signaling requires further studies in vivo not least, in order to prevent toxicity.

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  • 22. Bergkvist, Johanna
    et al.
    Klawonn, Isabell
    Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
    Whitehouse, Martin J.
    Stockholm University, Faculty of Science, Department of Geological Sciences. Swedish Museum of Natural History, Sweden.
    Lavik, Gaute
    Brüchert, Volker
    Stockholm University, Faculty of Science, Department of Geological Sciences.
    Ploug, Helle
    Turbulence simultaneously stimulates small-and large-scale CO2 sequestration by chain-forming diatoms in the sea2018In: Nature Communications, E-ISSN 2041-1723, Vol. 9, article id 3046Article in journal (Refereed)
    Abstract [en]

    Chain-forming diatoms are key CO2-fixing organisms in the ocean. Under turbulent conditions they form fast-sinking aggregates that are exported from the upper sunlit ocean to the ocean interior. A decade-old paradigm states that primary production in chain-forming diatoms is stimulated by turbulence. Yet, direct measurements of cell-specific primary production in individual field populations of chain-forming diatoms are poorly documented. Here we measured cell-specific carbon, nitrate and ammonium assimilation in two field populations of chain-forming diatoms (Skeletonema and Chaetoceros) at low-nutrient concentrations under still conditions and turbulent shear using secondary ion mass spectrometry combined with stable isotopic tracers and compared our data with those predicted by mass transfer theory. Turbulent shear significantly increases cell-specific C assimilation compared to still conditions in the cells/chains that also form fast-sinking, aggregates rich in carbon and ammonium. Thus, turbulence simultaneously stimulates small-scale biological CO2 assimilation and large-scale biogeochemical C and N cycles in the ocean.

  • 23.
    Bonath, Franziska
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Domingo-Prim, Judit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Tarbier, Marcel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Next-generation sequencing reveals two populations of damage- induced small RNAs at endogenous DNA double-strand breaksIn: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed)
  • 24. Bondarenko, Vasyl
    et al.
    Wells, Marta M.
    Chen, Qiang
    Tillman, Tommy S.
    Singewald, Kevin
    Lawless, Matthew J.
    Caporoso, Joel
    Brandon, Nicole
    Coleman, Jonathan A.
    Saxena, Sunil
    Lindahl, Erik
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab). KTH Royal Institute of Technology, Sweden.
    Xu, Yan
    Tang, Pei
    Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, article id 793Article in journal (Refereed)
    Abstract [en]

    The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes. 

  • 25. Brunetti, Dario
    et al.
    Torsvik, Janniche
    Dallabona, Cristina
    Teixeira, Pedro
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Sztromwasser, Pawel
    Fernandez-Vizarra, Erika
    Cerutti, Raffaele
    Reyes, Aurelio
    Preziuso, Carmela
    D'Amati, Giulia
    Baruffini, Enrico
    Goffrini, Paola
    Viscomi, Carlo
    Ferrero, Ileana
    Boman, Helge
    Telstad, Wenche
    Johansson, Stefan
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Knappskog, Per M.
    Zeviani, Massimo
    Bindoff, Laurence A.
    Defective PITRM1 mitochondrial peptidase is associated with A amyloidotic neurodegeneration2016In: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 8, no 3, p. 176-190Article in journal (Refereed)
    Abstract [en]

    Mitochondrial dysfunction and altered proteostasis are central features of neurodegenerative diseases. The pitrilysin metallopeptidase 1 (PITRM1) is a mitochondrial matrix enzyme, which digests oligopeptides, including the mitochondrial targeting sequences that are cleaved from proteins imported across the inner mitochondrial membrane and the mitochondrial fraction of amyloid beta (A). We identified two siblings carrying a homozygous PITRM1 missense mutation (c.548G>A, p.Arg183Gln) associated with an autosomal recessive, slowly progressive syndrome characterised by mental retardation, spinocerebellar ataxia, cognitive decline and psychosis. The pathogenicity of the mutation was tested invitro, in mutant fibroblasts and skeletal muscle, and in a yeast model. A Pitrm1(+/-) heterozygous mouse showed progressive ataxia associated with brain degenerative lesions, including accumulation of A-positive amyloid deposits. Our results show that PITRM1 is responsible for significant A degradation and that impairment of its activity results in A accumulation, thus providing a mechanistic demonstration of the mitochondrial involvement in amyloidotic neurodegeneration.

  • 26. Busayavalasa, Kiran
    et al.
    Chen, Xin
    Östlund Farrants, Ann-Kristin
    Stockholm University, Faculty of Science, The Wenner-Gren Institute , Cell Biology.
    Wagner, Nicole
    Sabri, Nafiseh
    The nup155 mediated organisation of inner nuclear membrane proteins is independent of nup155 anchoring to the metazoan nuclear pore complex2012In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 125, no 18, p. 4214-4218Article in journal (Refereed)
    Abstract [en]

    The nuclear envelope (NE), an important barrier between the nucleus and the cytoplasm, is composed of three structures: the outer nuclear membrane, which is continuous with the ER, the inner nuclear membrane (INM), which interfaces with chromatin, and nuclear pore complexes (NPCs), which are essential for the exchange of macromolecules between the two compartments. The NPC protein Nup155 has an evolutionarily conserved role in the metazoan NE formation; but the in vivo analysis of Nup155 has been severely hampered by the essential function of this protein in cell viability. Here, we take advantage of the hypomorphicity of RNAi systems and use a combination of protein binding and rescue assays to map the interaction sites of two neighbouring NPC proteins Nup93 and Nup53 on Nup155, and to define the requirements of these interactions in INM protein organization. We show that different parts of Drosophila Nup155 have distinct functions: the Nup155 beta-propeller anchors the protein to the NPC, whereas the alpha-solenoid part of Nup155 is essential for the correct localisation of INM proteins lamin-B receptor (LBR) and otefin. Using chromatin extracts from semisynchronized cells, we also provide evidence that the Nup155 alpha-solenoid has a chromatin-binding activity that is stronger at the end of mitosis. Our results argue that the role of Nup155 in INM protein localisation is not mediated through the NPC anchoring activity of the protein and suggest that regions other than Nup155 beta-propeller are necessary for the targeting of proteins to the INM.

  • 27. Byazrova, Maria
    et al.
    Gattinger, Pia
    Astakhova, Ekaterina
    Hofer, Gerhard
    Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK).
    Khaitov, Musa
    Filatov, Alexander
    Valenta, Rudolf
    Dissection of Antibody Responses of Gam-COVID-Vac-Vaccinated Subjects Suggests Involvement of Epitopes Outside RBD in SARS-CoV-2 Neutralization2023In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 6, article id 5104Article in journal (Refereed)
    Abstract [en]

    Millions of people have been vaccinated with Gam-COVID-Vac but fine specificities of induced antibodies have not been fully studied. Plasma from 12 naïve and 10 coronavirus disease 2019 (COVID-19) convalescent subjects was obtained before and after two immunizations with Gam-COVID-Vac. Antibody reactivity in the plasma samples (n = 44) was studied on a panel of micro-arrayed recombinant folded and unfolded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) proteins and 46 peptides spanning the spike protein (S) and by immunoglobulin G (IgG) subclass enzyme-linked immunosorbent assay (ELISA). The ability of Gam-COVID-Vac-induced antibodies to inhibit binding of the receptor-binding domain (RBD) to its receptor angiotensin converting enzyme 2 (ACE2) was investigated in a molecular interaction assay (MIA). The virus-neutralizing capacity of antibodies was studied by the pseudo-typed virus neutralization test (pVNT) for Wuhan-Hu-1 and Omicron. We found that Gam-COVID-Vac vaccination induced significant increases of IgG1 but not of other IgG subclasses against folded S, spike protein subunit 1 (S1), spike protein subunit 2 (S2), and RBD in a comparable manner in naïve and convalescent subjects. Virus neutralization was highly correlated with vaccination-induced antibodies specific for folded RBD and a novel peptide (i.e., peptide 12). Peptide 12 was located close to RBD in the N-terminal part of S1 and may potentially be involved in the transition of the pre- to post-fusion conformation of the spike protein. In summary, Gam-COVID-Vac vaccination induced S-specific IgG1 antibodies in naive and convalescent subjects in a comparable manner. Besides the antibodies specific for RBD, the antibodies induced against a peptide close to the N-terminus of RBD were also associated with virus-neutralization.

  • 28.
    Cannon, Barbara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    de Jong, Jasper M. A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Fischer, Alexander W.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Human brown adipose tissue: Classical brown rather than brite/beige?2020In: Experimental Physiology, ISSN 0958-0670, E-ISSN 1469-445X, Vol. 105, no 8, p. 1191-1200Article in journal (Refereed)
    Abstract [en]

    New Findings What is the topic of this review? It has been suggested that human brown adipose tissue (BAT) is more similar to the brite/beige adipose tissue of mice than to classical BAT of mice. The basis of this is discussed in relationship to the physiological conditions of standard experimental mice.

  • 29.
    Cannon, Barbara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A PERKy way to make mitochondrial cristae2021In: Trends in endocrinology and metabolism, ISSN 1043-2760, E-ISSN 1879-3061, Vol. 32, no 7, p. 417-419Article, review/survey (Refereed)
    Abstract [en]

    PERK protein, that is canonically associated with the response to endoplasmic reticulum stress, may be acquiring a new role as a regulator of the growth of mitochondrial cristae. This role is pertinent not only to the recruitment of brown adipose tissue thermogenic capacity but probably also to directing cristae formation in highly metabolically active organs such as the heart.

  • 30.
    Cannon, Barbara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    What Ignites UCP1?2017In: Cell Metabolism, ISSN 1550-4131, E-ISSN 1932-7420, Vol. 26, no 5, p. 697-698Article in journal (Other academic)
    Abstract [en]

    We thought we knew how the heat-producing uncoupling protein 1 in brown adipose tissue was activated: by fatty acids released upon lipid droplet breakdown in the brown adipocytes. However, two studies in this issue (Schreiber et al., 2017; Shin et al., 2017) imply that this classical model may not be valid: heat can be produced in brown fat without intracellular lipolysis.

  • 31. Carreras-Badosa, Gemma
    et al.
    Maslovskaja, Julia
    Periyasamy, Kapilraj
    Urgard, Egon
    Padari, Kärt
    Vaher, Helen
    Tserel, Liina
    Gestin, Maxime
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kisand, Kai
    Arukuusk, Piret
    Lou, Chenguang
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Tartu, Estonia.
    Wengel, Jesper
    Pooga, Margus
    Rebane, Ana
    NickFect type of cell-penetrating peptides present enhanced efficiency for microRNA-146a delivery into dendritic cells and during skin inflammation2020In: Biomaterials, ISSN 0142-9612, E-ISSN 1878-5905, Vol. 262, article id 120316Article in journal (Refereed)
    Abstract [en]

    MicroRNAs (miRNAs) are post-transcriptional gene expression regulators with potential therapeutic applications. miR-146a is a negative regulator of inflammatory processes in both tissue-resident and specialized immune cells and may therefore have therapeutic effect in inflammatory skin diseases. PepFect (PF) and NickFect (NF) type of cell-penetrating peptides (CPPs) have previously been shown to deliver miRNA mimics and/or siRNAs into cell cultures and in vivo. Here, we first demonstrate that selected PF- and NF-type of CPPs support delivery of fluorescent labelled miRNA mimics into keratinocytes (KCs) and dendritic cells (DCs). Second, we show that both PF- and NF-miR-146a nanocomplexes were equally effective in KCs, while NFs were more efficient in DCs as assessed by downregulation of miR-146a-influenced genes. None of miRNA nanocomplexes with the tested CPPs influenced the viability of KCs and DCs nor caused activation of DCs according to CD86 and CD83 markers. Transmission electron microscopy analysis with Nanogold-labelled miR-146a mimics and assessment of endocytic trafficking pathways revealed endocytosis as an active route of delivery in both KCs and DCs for all tested CPPs. However, consistent with the higher efficiency, NF-delivered miR-146a was detected more often outside endosomes in DCs. Finally, pre-injection of NF71:miR-146a nanocomplexes was confirmed to suppress inflammatory responses in a mouse model of irritant contact dermatitis as shown by reduced ear swelling response and downregulation of pro-inflammatory cytokines, including IL-6, IL-1 beta, IL-33 and TNF-alpha. In conclusion, NF71 efficiently delivers miRNA mimics into KCs as well as DCs, and therefore may have advantage in therapeutic delivery of miRNAs in case of inflammatory skin diseases.

  • 32. Cavalieri, Riccardo
    et al.
    Klein Hazebroek, Marlou
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cotrim, Camila A.
    Lee, Yang
    Kunji, Edmund R. S.
    Jastroch, Martin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Keipert, Susanne
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Crichton, Paul G.
    Activating ligands of Uncoupling protein 1 identified by rapid membrane protein thermostability shift analysis2022In: Molecular Metabolism, ISSN 2212-8778, Vol. 62, article id 101526Article in journal (Refereed)
    Abstract [en]

    Objective: Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1.

    Methods: We assessed ligand binding to purified UCP1 by protein thermostability shift analysis, which unlike many conventional approaches can inform on the binding of hydrophobic ligands to membrane proteins. A detailed activator interaction analysis and screening approach was carried out, supported by investigations of UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1 expression-controlled cell lines.

    Results: We reveal that fatty acids and other activators influence UCP1 through a specific destabilising interaction, behaving as transport substrates that shift the protein to a less stable conformation of a transport cycle. Through the detection of specific stability shifts in screens, we identify novel activators, including the over-the-counter drug ibuprofen, where ligand analysis indicates that UCP1 has a relatively wide structural specificity for interacting molecules. Ibuprofen successfully induced UCP1 activity in liposomes, isolated brown fat mitochondria and UCP1-expressing HEK293 cells but not in cultured brown adipocytes, suggesting drug delivery differs in each cell type.

    Conclusions: These findings clarify the nature of the activator-UCP1 interaction and demonstrate that the targeting of UCP1 in cells by approved drugs is in principle achievable as a therapeutic avenue, but requires variants with more effective delivery in brown adipocytes.

  • 33. Cedernaes, Jonathan
    et al.
    Schonke, Milena
    Orzechowski Westholm, Jakub
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Mi, Jia
    Chibalin, Alexander
    Voisin, Sarah
    Osler, Megan
    Vogel, Heike
    Hornaeus, Katarina
    Dickson, Suzanne L.
    Lind, Sara Bergstrom
    Bergquist, Jonas
    Schioth, Helgi B.
    Zierath, Juleen R.
    Benedict, Christian
    Acute sleep loss results in tissue-specific alterations in genome-wide DNA methylation state and metabolic fuel utilization in humans2018In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 8, article id eaar8590Article in journal (Refereed)
    Abstract [en]

    Curtailed sleep promotes weight gain and loss of lean mass in humans, although the underlying molecular mechanisms are poorly understood. We investigated the genomic and physiological impact of acute sleep loss in peripheral tissues by obtaining adipose tissue and skeletal muscle after one night of sleep loss and after one full night of sleep. We find that acute sleep loss alters genome-wide DNA methylation in adipose tissue, and unbiased transcriptome-, protein-, and metabolite-level analyses also reveal highly tissue-specific changes that are partially reflected by altered metabolite levels in blood. We observe transcriptomic signatures of inflammation in both tissues following acute sleep loss, but changes involving the circadian clock are evident only in skeletal muscle, and we uncover molecular signatures suggestive of muscle breakdown that contrast with an anabolic adipose tissue signature. Our findings provide insight into how disruption of sleep and circadian rhythms may promote weight gain and sarcopenia.

  • 34. Cerrato, Carmine Pasquale
    et al.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Tartu, Estonia.
    An update on cell-penetrating peptides with intracellular organelle targeting2022In: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 19, no 2, p. 133-146Article, review/survey (Refereed)
    Abstract [en]

    Introduction Cell-penetrating peptide (CPP) technologies represent an important strategy to address drug delivery to specific intracellular compartments by covalent conjugation to targeting sequences, potentially enabling strategies to combat most diseases.

    Areas covered This updated review article provides an overview of current intracellular organelle targeting by CPP. The targeting strategies of CPP and CPP/cargo complexes to specific cells or intracellular organelles are summarized, and the review provides an update on the current data for their pharmacological and therapeutical applications.

    Expert opinion Targeted drug delivery is moving from the level of tissue or specific pathogenic cell to the level of specific organelle that is the target of the drug, an important aspect in drug design and development. Organelle-targeted drug delivery results in improved efficacy, ability to control mode of action, reduction of undesired toxicities and side effects, and the possibility to overcome drug resistance mechanisms.

  • 35.
    Chiaka Akuwudike, Pamela
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Cellular effects of ionizing radiation: Relevant for understanding cancer risk after medical and environmental radiation exposures2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Radiation-induced cancers are stochastic and delayed effects of exposure to ionizing radiation. The dose-response relationship for radiation-induced cancers at both low dose/low dose rates and high doses (doses encountered during radiotherapy) remains unclear. Uncertainties observed in epidemiological studies at low doses and dose rates hamper cancer risk estimation at this dose level. Assessing dose-response relationships for radiotherapy-induced cancers is also complicated due to the inherent difficulty in assessing the doses absorbed by tissues at the site of tumours. In addition, the modulatory effect of chemotherapy on the incidence of radiotherapy-induced cancer risk has been debated. Although included as a modifying factor of the incidence of radiotherapy-induced cancers, results from epidemiological studies do not provide sufficient evidence to support this claim. This thesis summarizes studies conducted to improve the understanding of the association of cancer incidence and radiation dose at clinically relevant (high) doses, low doses and low dose rates, as well as the modulatory role of platinum-based chemotherapy on radiation-induced carcinogenesis. 

    In Paper I, we investigated the competitive relationship between cell killing and the accumulation of DNA damage and genomic instability using two normal cell types (VH10 fibroblasts and AHH-1 lymphoblasts). Dose fractionation schemes were designed based on the cell growth characteristics of each cell type. Cells were irradiated at 0.25, 0.5, 1.0, or 2 Gy per fraction, representing the various dose levels within a radiation field, to simulate the heterogeneous dose distribution across normal tissue during radiotherapy. Following fractionated radiation exposure, the effects on cell growth, cell survival, radiosensitivity, and the accumulation of residual DNA damage and genomic instability were analyzed as a function of dose per fraction and the total absorbed dose. The accumulation of DNA damage and markers of genomic instability associated with DNA damage depended on cell type-specific factors.

    In Paper II, we investigated the modulatory effects of combining cisplatin and radiation on the accumulation of micronuclei (a biomarker of DNA damage and carcinogenesis) in peripheral blood lymphocytes of patients receiving treatment for gynaecological cancers. We also determined the modulatory effects of the combination of both agents on cell death and cell proliferation, by scoring the frequency of apoptotic and binucleated cells. We compared the frequency of these markers between patients receiving treatment with radiotherapy alone and a combination of cisplatin and radiotherapy. There was a decline in the frequency of micronuclei in patients receiving a combination of cisplatin and radiotherapy.

    We conducted in vitro experiments in Paper III using AHH-1 and VH10 cells. We investigated the effects of the concurrent combination of cisplatin treatment and multifractionated radiation exposure at 1 Gy per fraction on cell growth, cell survival, cell death, changes in radiosensitivity, accumulation of DNA damage, and other markers of genomic instability as well as the expression of cancer stem cell markers. We also investigated the interaction between cisplatin and radiation exposure in our schedule. The concurrent combination of cisplatin and radiation did not increase the accumulation of markers of genomic instability.

    In Paper IV, we investigated the short and long-term effects of radiation exposure at low doses and low dose rates on global gene expression, cell growth and cell survival of VH10 fibroblasts to identify unique dose rate signatures that could be useful biomarkers in determining if the application of DDREF is accurate. Except for the differential expression of DMXL2, the long-term effects of LDLDR exposure on global gene expression, cell growth and cell survival of VH10 fibroblasts were negligible. These results suggest that the accumulation of DNA damage and other markers of genomic instability is regulated by cell type-specific factors at these dose levels.

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  • 36. Chiurchiu, Valerio
    et al.
    Leuti, Alessandro
    Dalli, Jesmond
    Jacobsson, Anders
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Battistini, Luca
    Maccarrone, Mauro
    Serhan, Charles N.
    Proresolving lipid mediators resolvin D1, resolvin D2, and maresin 1 are critical in modulating T cell responses2016In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 8, no 353, article id 353ra111Article in journal (Refereed)
    Abstract [en]

    Resolution of inflammation is a finely regulated process mediated by specialized proresolving lipid mediators (SPMs), including docosahexaenoic acid (DHA)-derived resolvins and maresins. The immunomodulatory role of SPMs in adaptive immune cells is of interest. We report that D-series resolvins (resolvin D1 and resolvin D2) and maresin 1 modulate adaptive immune responses in human peripheral blood lymphocytes. These lipid mediators reduce cytokine production by activated CD8(+) T cells and CD4(+) T helper 1 (T(H)1) and T(H)17 cells but do not modulate T cell inhibitory receptors or abrogate their capacity to proliferate. Moreover, these SPMs prevented naive CD4(+) T cell differentiation into T(H)1 and T(H)17 by down-regulating their signature transcription factors, T-bet and Rorc, in a mechanism mediated by the GPR32 and ALX/FPR2 receptors; they concomitantly enhanced de novo generation and function of Foxp3(+) regulatory T (T-reg) cells via the GPR32 receptor. These results were also supported in vivo in a mouse deficient for DHA synthesis (Elovl2(-/-)) that showed an increase in T(H)1/T(H)17 cells and a decrease in T-reg cells compared to wild-type mice. Additionally, either DHA supplementation in Elovl2(-/-) mice or in vivo administration of resolvin D1 significantly reduced cytokine production upon specific stimulation of T cells. These findings demonstrate actions of specific SPMs on adaptive immunity and provide a new avenue for SPM-based approaches to modulate chronic inflammation.

  • 37. Coenen-Stass, Anna M. L.
    et al.
    Sork, Helena
    Gatto, Sole
    Godfrey, Caroline
    Bhomra, Amarjit
    Krjutskov, Kaarel
    Hart, Jonathan R.
    Westholm, Jakub O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    O'Donovan, Liz
    Roos, Andreas
    Lochmueller, Hanns
    Puri, Pier Lorenzo
    EL Andaloussi, Samir
    Wood, Matthew J. A.
    Roberts, Thomas C.
    Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD2018In: Molecular Therapy Nucleic Acids, E-ISSN 2162-2531, Vol. 13, p. 1-15Article in journal (Refereed)
    Abstract [en]

    Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.

  • 38. Darai-Ramqvist, Eva
    et al.
    Nilsonne, Gustav
    Stockholm University, Faculty of Social Sciences, Stress Research Institute. Karolinska Institutet, Sweden.
    Flores-Staino, Carmen
    Hjerpe, Anders
    Dobra, Katalin
    Microenvironment-dependent phenotypic changes in a SCID mouse model for malignant mesothelioma2013In: Frontiers in Oncology, E-ISSN 2234-943X, Vol. 3, article id 203Article in journal (Refereed)
    Abstract [en]

    Background and Aims: Malignant mesothelioma is an aggressive, therapy-resistant tumor. Mesothelioma cells may assume an epithelioid or a sarcomatoid phenotype, and presence of sarcomatoid cells predicts poor prognosis. In this study, we investigated differentiation of mesothelioma cells in a xenograft model, where mesothelioma cells of both phenotypes were induced to form tumors in severe combined immunodeficiency mice.

    Methods: Xenografts were established and thoroughly characterized using a comprehensive immunohistochemical panel, array comparative genomic hybridization (aCGH) of chromosome 3, fluorescent in situ hybridization, and electron microscopy.

    Results: Epithelioid and sarcomatoid cells gave rise to xenografts of similar epithelioid morphology. While sarcomatoid-derived xenografts had higher growth rates, the morphology and expression of differentiation-related markers was similar between xenografts derived from both phenotypes. aCGH showed a convergent genotype for both xenografts, resembling the original aggressive sarcomatoid cell sub-line.

    Conclusion: Human mesothelioma xenografts from sarcomatoid and epithelioid phenotypes converged to a similar differentiation state, and genetic analyses suggested that clonal selection in the mouse microenvironment was a major contributing factor. This thoroughly characterized animal model can be used for further studies of molecular events underlying tumor cell differentiation.

  • 39.
    de Jong, Jasper M. A.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Yale School of Medicine, USA.
    Sun, Wenfei
    Pires, Nuno D.
    Frontini, Andrea
    Balaz, Miroslav
    Jespersen, Naja Z.
    Feizi, Amir
    Petrovic, Katarina
    Fischer, Alexander W.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. University Medical Center Hamburg-Eppendorf, Germany; Chan School of Public Health, USA; Harvard Medical School, USA.
    Bokhari, Muhammad Hamza
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Niemi, Tarja
    Nuutila, Pirjo
    Cinti, Saverio
    Nielsen, Soren
    Scheele, Camilla
    Virtanen, Kirsi
    Cannon, Barbara
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nedergaard, Jan
    Wolfrum, Christian
    Petrovic, Natasa
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Human brown adipose tissue is phenocopied by classical brown adipose tissue in physiologically humanized mice2019In: Nature Metabolism, E-ISSN 2522-5812, Vol. 1, no 8, p. 830-843Article in journal (Refereed)
    Abstract [en]

    Human and rodent brown adipose tissues (BAT) appear morphologically and molecularly different. Here we compare human BAT with both classical brown and brite/beige adipose tissues of 'physiologically humanized' mice: middle-aged mice living under conditions approaching human thermal and nutritional conditions, that is, prolonged exposure to thermoneutral temperature (approximately 30 degrees C) and to an energy-rich (high-fat, high-sugar) diet. We find that the morphological, cellular and molecular characteristics (both marker and adipose-selective gene expression) of classical brown fat, but not of brite/beige fat, of these physiologically humanized mice are notably similar to human BAT. We also demonstrate, both in silico and experimentally, that in physiologically humanized mice only classical BAT possesses a high thermogenic potential. These observations suggest that classical rodent BAT is the tissue of choice for translational studies aimed at recruiting human BAT to counteract the development of obesity and its comorbidities.

  • 40.
    Diessl, Jutta
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Metal homeostasis as critical determinant for cellular fitness2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Metals play a crucial role in cellular biology. Bulk and trace metals such as calcium and manganese regulate a plethora of cellular processes ranging from signaling and oxidative stress to proteostasis and energy metabolism. Fine-tuning metal levels and distribution safeguards all forms of life from compromised cellular fitness and cell death elicited by metal deficiency or overload. However, the underlying molecular mechanisms eventually leading to cellular demise remain elusive. In this thesis, we studied the fundamental impact of disrupted metal homeostasis on cellular survival focusing on mitochondrial and lysosomal processes in Saccharomyces cerevisiae and Drosophila melanogaster. In Paper I, we establish Coenzyme Q (CoQ) biosynthesis in mitochondria as the prime target of cellular manganese overload and propose a molecular mechanism underlying manganese toxicity. Combining proteomics, genome-wide screening and comprehensive metal analyses, we identify mismetallation of the di-iron hydroxylase Coq7, an enzyme of CoQ biosynthesis, as cause for the CoQ deficiency upon manganese overload. Overexpression of Coq7 not only restored CoQ-mediated electron transport through the respiratory chain but also prevented age-associated death. Expanding from trace to bulk metals, we further assessed the impact of disrupted calcium and manganese homeostasis on cellular survival. In Paper II, we created a fluorescence-based reporter system for the Ca2+/calmodulin-dependent phosphatase calcineurin, a nexus for cell stress-induced signaling. Combining our reporters with a live/dead staining allows for quantification of acute and chronic changes in calcium signaling in living, unperturbed cells. In Paper III, we elucidate the impact of nutritional regimes known to improve cellular survival on cells compromised in the handling of calcium and manganese due to the absence of Pmr1, a Ca2+/Mn2+ ATPase of the secretory pathway. We demonstrate that caloric restriction prevents manganese-induced disruption of mitochondrial energy metabolism and improves survival independent of calcineurin activity and CoQ biosynthesis. In Papers IV and V, we studied the interplay of metal levels and calcium signaling in the context of neurodegeneration and report that calcineurin stimulates lysosomal proteolysis, thereby preventing proteotoxicity in yeast and Drosophila models for Parkinson’s disease. Collectively, our results provide new insights into the consequences of disrupted metal homeostasis for cellular fitness and unravel a novel link between manganese overload, mitochondrial bioenergetics and CoQ biosynthesis conserved across species.

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  • 41.
    Diessl, Jutta
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Berndtsson, Jens
    Broeskamp, Filomena
    Habernig, Lukas
    Kohler, Verena
    Vazquez-Calvo, Carmela
    Nandy, Arpita
    Peselj, Carlotta
    Drobysheva, Sofia Polina
    Pelosi, Ludovic
    Vögtle, Nora F.
    Pierrel, Fabien
    Ott, Martin
    Büttner, Sabrina
    Manganese overload disrupts mitochondrial energy metabolism via inhibition of Coenzyme Q biosynthesisManuscript (preprint) (Other academic)
  • 42.
    Diessl, Jutta
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Habernig, Lukas
    Prado Morales, Carles
    Pierrel, Fabien
    Büttner, Sabrina
    Caloric restriction prevents manganese-induced disruption of mitochondrial bioenergeticsManuscript (preprint) (Other academic)
  • 43. Divakaruni, Ajit S.
    et al.
    Jastroch, Martin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    A practical guide for the analysis, standardization and interpretation of oxygen consumption measurements2022In: Nature Metabolism, E-ISSN 2522-5812, Vol. 4, no 8, p. 978-994Article in journal (Refereed)
    Abstract [en]

    Measurement of oxygen consumption is a powerful and uniquely informative experimental technique. It can help identify mitochondrial mechanisms of action following pharmacologic and genetic interventions, and characterize energy metabolism in physiology and disease. The conceptual and practical benefits of respirometry have made it a frontline technique to understand how mitochondrial function can interface with—and in some cases control—cell physiology. Nonetheless, an appreciation of the complexity and challenges involved with such measurements is required to avoid common experimental and analytical pitfalls. Here we provide a practical guide to oxygen consumption measurements covering the selection of experimental models and instrumentation, as well as recommendations for the collection, interpretation and normalization of data. These guidelines are provided with the intention of aiding experimental design and enhancing the overall reputability, transparency and reliability of oxygen consumption measurements.

  • 44.
    Domingo Prim, Judit
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    The RNA exosome and the maintenance of genome integrity2018Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The RNA exosome is a ribonucleolytic complex that acts on different RNA substrates and plays important roles in RNA metabolism. In recent years, the synthesis and the processing of RNA have been directly linked to the integrity of the genome. RNAs can either be the responsible for genomic instability or, on the contrary, can participate in the DNA damage response. Damage-induced RNAs (diRNAs) are short non-coding RNAs that have been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination. The implication of specialized RNAs in DNA damage and repair led us to investigate whether the exosome was involved in DNA repair.

    In Paper I, we have shown by fluorescence microscopy and chromatin immunoprecipitation that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DSBs in Drosophila and human cells. Depletion of this subunit or overexpression of a catalytically inactive mutant makes the cells more sensitive to radiation and unable to recruit the homologous recombination factor RAD51 to DSBs, which is consistent with RRP6/EXOSC10 playing a role in homologous recombination, both in insect and mammalian cells. The results obtained with the RRP6 inactive mutant also suggest that the ribonucleolytic activity of RRP6 is required for DNA repair. However, the mechanisms by which RNAs and the exosome are implicated in DNA repair need to be further investigated.

    In Paper II, we describe how transcription of DSB-flanking sequences by RNA polymerase II gives rise to damage-induced long non-coding RNAs that are processed into diRNAs. The direct detection of diRNAs had been elusive and their existence had been questioned, but our results show that damage-induced transcription and diRNA production occur at DSBs in endogenous, repetitive genomic sequences in mammalian cells. However, our exhaustive next-generation sequencing failed to detect diRNAs derived from DSBs in unique sequences. The diRNAs produced at repetitive loci bind to Argonaute and belong to two different subpopulations. One of them is Dicer-dependent and has a length of 21-22 nucleotides. The other one is not yet well characterized and is probably composed of degradation products from other ribonucleases.

    Finally, in Paper III, we have demonstrated that EXOSC10 is one of the ribonucleases involved in RNA degradation at DSBs. By strand-specific quantitative PCR and RNA-seq, we show that the levels of diRNA precursors and diRNAs are increased in the absence of EXOSC10. Moreover, EXOSC10-depleted cells fail to recruit RPA to DSBs, and this defect is restored by RNase A digestion. Depletion of EXOSC10 also results in extended DNA resected tracks, as shown by both single-molecule analysis of resected tracks and quantitative amplification of single-stranded DNA. These results suggest that EXOSC10 is involved in RNA degradation at DSBs to allow RPA recruitment and regulated resection.

    The work presented in this thesis supports the conclusion that damage-induced RNAs are synthesized de novo by RNA polymerase II at DSBs in mammalian cells. In repetitive genomic loci, these RNAs are processed into diRNAs that bind Argonaute. Regardless of whether diRNAs are functional or not, their precursors have to be degraded. The main function of the exosome, and more specifically EXOSC10, in the maintenance of the integrity of the genome is to degrade these transcripts in order to allow faithful repair of DNA double-strand breaks by homologous recombination.

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  • 45.
    Domingo-Prim, Judit
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Endara-Coll, Martín
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Bonath, Franziska
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Jimeno, Sonia
    Friedländer, Marc
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Huertas, Pablo
    Visa, Neus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    EXOSC10 is required for RPA assembly and controlled DNA resection at DNA dobule-strand breaksManuscript (preprint) (Other academic)
  • 46.
    Dondalska, Aleksandra
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Axberg Pålsson, Sandra
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Spetz, Anna-Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Is There a Role for Immunoregulatory and Antiviral Oligonucleotides Acting in the Extracellular Space? A Review and Hypothesis2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 23, article id 14593Article, review/survey (Refereed)
    Abstract [en]

    Here, we link approved and emerging nucleic acid-based therapies with the expanding universe of small non-coding RNAs (sncRNAs) and the innate immune responses that sense oligonucleotides taken up into endosomes. The Toll-like receptors (TLRs) 3, 7, 8, and 9 are located in endosomes and can detect nucleic acids taken up through endocytic routes. These receptors are key triggers in the defense against viruses and/or bacterial infections, yet they also constitute an Achilles heel towards the discrimination between self- and pathogenic nucleic acids. The compartmentalization of nucleic acids and the activity of nucleases are key components in avoiding autoimmune reactions against nucleic acids, but we still lack knowledge on the plethora of nucleic acids that might be released into the extracellular space upon infections, inflammation, and other stress responses involving increased cell death. We review recent findings that a set of single-stranded oligonucleotides (length of 25–40 nucleotides (nt)) can temporarily block ligands destined for endosomes expressing TLRs in human monocyte-derived dendritic cells. We discuss knowledge gaps and highlight the existence of a pool of RNA with an approximate length of 30–40 nt that may still have unappreciated regulatory functions in physiology and in the defense against viruses as gatekeepers of endosomal uptake through certain routes.

  • 47.
    Dziedziech, Alexis
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Timing Matters: Wounding and entomopathogenic nematode infection kinetics2021Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Over time, insects have developed complex strategies to defend themselves against presenting threats. However, in the evolutionary arms race of survival, pathogens have adapted to quickly overcome the immune response mounted by the host. In this thesis, we assess how quickly entomopathogenic nematodes (EPNs) can overcome the host, Drosophila melanogaster. We then look at the clotting reaction at a hypothetical point of entry for the nematode and bring resolution to the order of protein interaction focusing on three proteins important in the anti-nematode defense. Finally, we look closer into detail at how crystal cells secrete one of those proteins, prophenoloxidase (PPOII) using a mode of programmed cell death. 

    (Paper I) In the course of EPN infection, little was known about how quickly the worms can overcome the host immune system. Here we found that after penetrating the host, EPNs cause septicemia within 4 to 6 hours. (Paper II) Three proteins, Glutactin (Glt), Transglutaminase (Tg), and PPOII have been found to be important in the anti-nematode response. Here we created GFP-tagged fly constructs to follow their role in clot formation. In early clot formation, Tg was immediately secreted from hemocytes though it was localized around the cell membrane, Glt then entered clot fibers followed by PPOII which acted in late clot formation. (Paper III) Here we looked closer into Tg and PPOII secretion variability. PPOII from immature, but not mature crystal cells colocalized with a membrane marker. Tg, when driven with a pan tissue driver, was found located in clotting fibers, in contrast with paper II. (Paper IV) In an in vivo immune scenario, crystal cells were recruited to the wound site and burst rapidly in a caspase-dependent manner. We demonstrate that the mode of programmed cell death, pyroptosis, exists in Drosophila by way of convergent evolution.

    This thesis brings to light the variation found within the infection process for EPNs as well as the clotting response based on larval age, tissue type, and the maturity of a single cell type. Timing in each of these immune scenarios can give very different indications about the kind of immune response mounted and even the role of an individual cell.

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  • 48. Edfeldt, Gabriella
    et al.
    Kaldhusdal, Vilde
    Czarnewski, Paulo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Bradley, Frideborg
    Bergström, Sofia
    Lajoie, Julie
    Xu, Jiawu
    Månberg, Anna
    Kimani, Joshua
    Oyugi, Julius
    Nilsson, Peter
    Tjernlund, Annelie
    Fowke, Keith R.
    Kwon, Douglas S.
    Broliden, Kristina
    Distinct cervical tissue-adherent and luminal microbiome communities correlate with mucosal host gene expression and protein levels in Kenyan sex workers2023In: Microbiome, E-ISSN 2049-2618, Vol. 11, article id 67Article in journal (Refereed)
    Abstract [en]

    Background The majority of studies characterizing female genital tract microbiota have focused on luminal organisms, while the presence and impact of tissue-adherent ectocervical microbiota remain incompletely understood. Studies of luminal and tissue-associated bacteria in the gastrointestinal tract suggest that these communities may have distinct roles in health and disease. Here, we performed a multi-omics characterization of paired luminal and tissue samples collected from a cohort of Kenyan female sex workers.

    Results We identified a tissue-adherent bacterial microbiome, with a higher alpha diversity than the luminal microbiome, in which dominant genera overall included Gardnerella and Lactobacillus, followed by Prevotella, Atopobium, and Sneathia. About half of the L. iners-dominated luminal samples had a corresponding Gardnerella-dominated tissue microbiome. Broadly, the tissue-adherent microbiome was associated with fewer differentially expressed host genes than the luminal microbiome. Gene set enrichment analysis revealed that L. crispatus-dominated tissue-adherent communities were associated with protein translation and antimicrobial activity, whereas a highly diverse microbial community was associated with epithelial remodeling and pro-inflammatory pathways. Tissue-adherent communities dominated by L. iners and Gardnerella were associated with lower host transcriptional activity. Tissue-adherent microbiomes dominated by Lactobacillus and Gardnerella correlated with host protein profiles associated with epithelial barrier stability, although with a more pro-inflammatory profile for the Gardnerella-dominated microbiome group. Tissue samples with a highly diverse composition had a protein profile representing cell proliferation and pro-inflammatory activity.

    Conclusion We identified ectocervical tissue-adherent bacterial communities in all study participants of a female sex worker cohort. These communities were distinct from cervicovaginal luminal microbiota in a significant proportion of individuals. We further revealed that bacterial communities at both sites correlated with distinct host gene expression and protein levels. The tissue-adherent bacterial community could possibly act as a reservoir that seed the lumen with less optimal, non-Lactobacillus, bacteria.

  • 49.
    Ekberg, Monica
    Stockholm University.
    Radical transfer in ribonucleotide reductase from Escherichia coli2000Doctoral thesis, comprehensive summary (Other academic)
  • 50.
    EL Andaloussi, Samir
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Lehto, Taavi
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Lundin, Per
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Application of PepFect peptides for the delivery of splice-correcting oligonucleotides2011In: Cell-penetrating peptides: Methods and Protocols, New York: Humana Press, 2011, p. 361-373Chapter in book (Other academic)
    Abstract [en]

    One oligonucleotide-based approach that appear very promising for the treatment of different genetic disorders are based on so-called splice-correcting oligonucleotides (SCOs) that are exploited to manipulate splicing patterns. In order to increase the bioavailability, cell-penetrating peptides (CPPs) have readily been covalently conjugated to SCOs to facilitate cellular internalization. While being a successful strategy for the delivery of uncharged oligonucleotides (ONs), it is extremely difficult to generate covalent conjugates between commonly used negatively charged ON analogs and cationic CPPs. Furthermore, high concentrations of ONs in the micromolar range are often needed to obtain biological responses, most likely as a result of endosomal entrapment of material. Therefore, exploring other vectorization methods using CPPs with endosomolytic properties are highly desired. A method of using stearyl modified CPP (i.e., TP10) analogs, named PepFect3 and PepFect4, are being described for the transfection of antisense SCOs using a simple one-step co-incubation procedure. These peptides form complexes with SCOs and efficiently promote cellular uptake by facilitating endosomal escape. This chapter describes the methods of how to form and characterize these nanoparticles and the cellular assay used to address the delivery.

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