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  • 101.
    Björkholm, Patrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Daniluk, Pawel
    Kryshtafovych, Andriy
    Fidelis, Krzysztof
    Andersson, Robin
    Hvidsten, Torgeir R.
    Using multi-data hidden Markov models trained on local neighborhoods of protein structure to predict residue-residue contacts2009Ingår i: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 25, nr 10, s. 1264-1270Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Motivation: Correct prediction of residue-residue contacts in proteins that lack good templates with known structure would take ab initio protein structure prediction a large step forward. The lack of correct contacts, and in particular long-range contacts, is considered the main reason why these methods often fail. Results: We propose a novel hidden Markov model (HMM)based method for predicting residue-residue contacts from protein sequences using as training data homologous sequences, predicted secondary structure and a library of local neighborhoods (local descriptors of protein structure). The library consists of recurring structural entities incorporating short-, medium- and long-range interactions and is general enough to reassemble the cores of nearly all proteins in the PDB. The method is tested on an external test set of 606 domains with no significant sequence similarity to the training set as well as 151 domains with SCOP folds not present in the training set. Considering the top 0.2 . L predictions (L = sequence length), our HMMs obtained an accuracy of 22.8% for long-range interactions in new fold targets, and an average accuracy of 28.6% for long-, medium- and short- range contacts. This is a significant performance increase over currently available methods when comparing against results published in the literature.

  • 102.
    Björkholm, Patrik
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ernst, Andreas M.
    Hacke, Moritz
    Wieland, Felix
    Bruegger, Britta
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Identification of novel sphingolipid-binding motifs in mammalian membrane proteins2014Ingår i: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, nr 8, s. 2066-2070Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.

  • 103. Blackshear, Alice
    et al.
    Yamamoto, Mihoko
    Anderson, Brenda J.
    Holmes, Philip V.
    Lundström, Linda
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Robinson, John K.
    Intracerebroventricular administration of galanin or galanin receptor subtype 1 agonist M617 induces c-Fos activation in central amygdala and dorsomedial hypothalamus2007Ingår i: Peptides, ISSN 0196-9781, E-ISSN 1873-5169, Vol. 28, nr 5, s. 1120-1124Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The neuropeptide galanin and galanin receptors are widespread throughout cortical, limbic and midbrain areas implicated in reward, learning/memory, pain, drinking and feeding. While many studies have shown that galanin produces a variety of presynaptic and postsynaptic responses, work studying the effects of galanin on neural activation is limited. The present study examined patterns of c-Fos immunoreactivity resulting from intracerebro-ventricular administration of galanin versus saline injection in awake rats. An initial comprehensive qualitative survey was conducted to identify regions of high c-Fos expression followed up with quantitative analysis. Galanin induced a significant increase in c-Fos levels relative to saline-treated controls in dorsomedial hypothalamus and in the central nucleus of the amygdala. This pattern of activation was also produced by galanin receptor type 1 agonist M617. The present findings confirm that galanin upregulates c-Fos activation in hypothalamic nuclei, and supports roles for galanin in central amygdala-mediated food intake, and Pavlovian conditioning.

  • 104.
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Can Reduction of NO to N2O in Cytochrome c Dependent Nitric Oxide Reductase Proceed through a Trans-Mechanism?2017Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, nr 1, s. 120-131Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    As part of microbial denitrification, NO is reduced to N2O in the membrane bound enzyme nitric oxide reductase, NOR The N N coupling occurs in the diiron binuclear active site, BNC, and different mechanisms for this reaction step have been suggested. Computational studies have supported a so-called cis:b(3)-mechanism, in which the hyponitrite product of the reductive N N bond formation coordinates with one nitrogen to the heme iron and with both oxygens to the non-heme iron in the BNC. In contrast, experimental results have been interpreted to support a so-called trans-mechanism, in which the hyponitrite intermediate coordinates with one nitrogen atom to each of the two iron ions. Hybrid density functional theory is used here to perform an extensive search for possible intermediates of the NO reduction in the cNOR enzyme. It is found that hyponitrite structures coordinating with their negatively charged oxygens to the positively charged iron ions are the most stable ones. The hyponitrite intermediate involved in the suggested trans-mechanism, which only coordinates with the nitrogens to the iron ions, is found to be prohibitively high in energy, leading to a too slow reaction, which should rule out this mechanism. Furthermore, intermediates binding one NO molecule to each iron ion in the BNC, which have been suggested to initiate the trans-mechanism, are found to be too high in energy to be observable, indicating that the experimentally observed electron paramagnetic resonance signals, taken to support such an iron-nitrosyl dimer intermediate, should be reinterpreted.

  • 105.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Siegbahn, Per E. M.
    Mechanism for N2O Generation in Bacterial Nitric Oxide Reductase: A Quantum Chemical Study2012Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, nr 25, s. 5173-5186Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The catalytic mechanism of reduction of NO to N2O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N2O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.

  • 106.
    Blomberg, Margareta R. A.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för organisk kemi.
    Why is the reduction of NO in cytochrome c dependent nitric oxide reductase (cNOR) not electrogenic?2013Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1827, nr 7, s. 826-833Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The membrane-bound enzyme cNOR (cytochrome c dependent nitric oxide reductase) catalyzes the reduction of NO in a non-electrogenic process. This is in contrast to the reduction of O-2 in cytochrome c oxidase (CcO), the other member of the heme-copper oxidase family, which stores energy by the generation of a membrane gradient. This difference between the two enzymes has not been understood, but it has been speculated to be of kinetic origin, since per electron the NO reduction is more exergonic than the O-2 reduction, and the energy should thus be enough for an electrogenic process. However, it has not been clear how and why electrogenicity, which mainly affects the thermodynamics, would slow down the very exergonic NO reduction. Quantum chemical calculations are used to construct a free energy profile for the catalytic reduction of NO in the active site of cNOR. The energy profile shows that the reduction of the NO molecules by the enzyme and the formation of N2O are very exergonic steps, making the rereduction of the enzyme endergonic and rate-limiting for the entire catalytic cycle. Therefore the NO reduction cannot be electrogenic, i.e. cannot take electrons and protons from the opposite sides of the membrane, since it would increase the endergonicity of the rereduction when the gradient is present, thereby increasing the rate-limiting barrier, and the reaction would become too slow. It also means that proton pumping coupled to electron transfer is not possible in cNOR In CcO the corresponding rereduction of the enzyme is very exergonic.

  • 107. Boban, Mirta
    et al.
    Ljungdahl, Per O.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Foisner, Roland
    Atypical Ubiquitylation in Yeast Targets Lysine-less Asi2 for Proteasomal Degradation2015Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 290, nr 4, s. 2489-2495Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Proteins are typically targeted for proteasomal degradation by the attachment of a polyubiquitin chain to epsilon-amino groups of lysine residues. Non-lysine ubiquitylation of proteasomal substrates has been considered an atypical and rare event limited to complex eukaryotes. Here we report that a fully functional lysine-less mutant of an inner nuclear membrane protein in yeast, Asi2, is polyubiquitylated and targeted for proteasomal degradation. Efficient degradation of lysine-free Asi2 requires E3-ligase Doa10 and E2 enzymes Ubc6 and Ubc7, components of the endoplasmic reticulum-associated degradation pathway. Together, our data suggest that non-lysine ubiquitylation may be more prevalent than currently considered.

  • 108.
    Boekel, Carolina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Integration and topology of membrane proteins2009Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Membrane proteins comprise around 20-30% of most proteomes. They play important roles in most biochemical pathways. All receptors and ion channels are membrane proteins, which make them attractive targets for drug design. Membrane proteins insert and fold co-translationally into the endoplasmic reticular membrane of eukaryotic cells. The protein-conducting channel that inserts the protein into the membrane is called Sec61 translocon, which is a hetero-oligomeric channel that allows transmembrane segments to insert laterally into the lipid bilayer. The focus of this thesis is how the translocon recognizes the transmembrane helices and integrates them into the membrane.

    We have investigated the sequence requirements for the translocon-mediated integration of a transmembrane α-helix into the ER by challenging the Sec61 translocon with designed polypeptide segments in an in vitro expression system that allows a quantitative assessment of membrane insertion efficiency. Our studies suggest that helices might interact with each other already during the membrane-insertion step, possibly forming helical hairpins that partition into the membrane as a single unit. Further, the insertion efficiency for Nin-Cout vs. Nout-Cin transmembrane helices and the integration efficiency of Alzheimer’s Aβ-peptide fragments has been investigated.

    Finally, detailed topology mapping was performed on two biologically interesting proteins with unknown topology, the human seipin protein and Drosophila melanogaster odorant receptor OR83b.

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  • 109. Boije, Henrik
    et al.
    Ring, Henrik
    Fard, Shahrzad Shirazi
    Grundberg, Ida
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab). Uppsala University .
    Hallbook, Finn
    Alternative Splicing of the Chromodomain Protein Morf4l1 Pre-mRNA Has Implications on Cell Differentiation in the Developing Chicken Retina2013Ingår i: Journal of Molecular Neuroscience, ISSN 0895-8696, E-ISSN 1559-1166, Vol. 51, nr 2, s. 615-628Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The proliferation, cell cycle exit and differentiation of progenitor cells are controlled by several different factors. The chromodomain protein mortality factor 4-like 1 (Morf4l1) has been ascribed a role in both proliferation and differentiation. Little attention has been given to the existence of alternative splice variants of the Morf4l1 mRNA, which encode two Morf41l isoforms: a short isoform (S-Morf4l1) with an intact chromodomain and a long isoform (L-Morf4l1) with an insertion in or in the vicinity of the chromodomain. The aim of this study was to investigate if this alternative splicing has a function during development. We analysed the temporal and spatial distribution of the two mRNAs and over-expressed both isoforms in the developing retina. The results showed that the S-Morf4l1 mRNA is developmentally regulated. Over-expression of S-Morf4l1 using a retrovirus vector produced a clear phenotype with an increase of early-born neurons: retinal ganglion cells, horizontal cells and cone photoreceptor cells. Over-expression of L-Morf4l1 did not produce any distinguishable phenotype. The over-expression of S-Morf4l1 but not L-Morf4l1 also increased apoptosis in the infected regions. Our results suggest that the two Morf4l1 isoforms have different functions during retinogenesis and that Morf4l1 functions are fine-tuned by developmentally regulated alternative splicing. The data also suggest that Morf4l1 contributes to the regulation of cell genesis in the retina.

  • 110.
    Bonath, Franziska
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Domingo-Prim, Judit
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Tarbier, Marcel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Friedländer, Marc R.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Visa, Neus
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Next-generation sequencing reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks2018Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, nr 22, s. 11869-11882Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Recent studies suggest that transcription takes place at DNA double-strand breaks (DSBs), that transcripts at DSBs are processed by Drosha and Dicer into damage-induced small RNAs (diRNAs), and that diRNAs are required for DNA repair. However, diRNAs have been mostly detected in reporter constructs or repetitive sequences, and their existence at endogenous loci has been questioned by recent reports. Using the homing endonuclease I-PpoI, we have investigated diRNA production in genetically unperturbed human and mouse cells. I-PpoI is an ideal tool to clarify the requirements for diRNA production because it induces DSBs in different types of loci: the repetitive 28S locus, unique genes and intergenic loci. We show by extensive sequencing that the rDNA locus produces substantial levels of diRNAs, whereas unique genic and intergenic loci do not. Further characterization of diRNAs emerging from the 28S locus reveals the existence of two diRNA subtypes. Surprisingly, Drosha and its partner DGCR8 are dispensable for diRNA production and only one diRNAs subtype depends on Dicer processing. Furthermore, we provide evidence that diRNAs are incorporated into Argonaute. Our findings provide direct evidence for diRNA production at endogenous loci in mammalian cells and give insights into RNA processing at DSBs.

  • 111. Borowski, Tomasz
    et al.
    Wojcik, Anna
    Milaczewska, Anna
    Georgiev, Valentin
    Blomberg, Margareta R. A.
    Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum. Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Siegbahn, Per E. M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Naturvetenskapliga fakulteten, Fysikum.
    The alkenyl migration mechanism catalyzed by extradiol dioxygenases: a hybrid dft study2012Ingår i: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 17, nr 6, s. 881-890Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    6-Hydroxymethyl-6-methylcyclohexa-2,4-dienone is a mechanistic probe which when incubated with an extradiol dioxygenase yields a 2-tropolone product. This observation was originally interpreted as evidence supporting a direct heterolytic 1,2-alkenyl migration mechanism for a ring expansion reaction catalyzed by this class of Fe(II)-dependent nonheme enzymes (Xin and Bugg in J Am Chem Soc 130:10422-10430, 2008). In the work reported in this contribution we used quantum chemical methods to test whether such a mechanism is energetically possible and we found that it is not, neither for the mechanistic probe nor for the native catalytic cycle intermediate. Models of increasing complexity were used to calculate energy barriers to the heterolytic 1,2-alkenyl migration and alternative radical mechanisms. It was found that the former involves substantially higher barriers than the latter. A tentative radical mechanism that accounts for the transformation of the probe substrate to 2-tropolone was also proposed, and it involves acceptable barriers.

  • 112.
    Braesch-Andersen, Sten
    Stockholms universitet.
    Studies of CD40, a growth factor receptor on B cells and certain carcinomas1991Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 113. Branca, Rui M. M.
    et al.
    Orre, Lukas M.
    Johansson, Henrik J.
    Granholm, Viktor
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Huss, Mikael
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Perez-Bercoff, Åsa
    Forshed, Jenny
    Käll, Lukas
    Lehtio, Janne
    HiRIEF LC-MSMS enables deep proteome coverage and unbiased proteogenomics2014Ingår i: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 11, nr 1, s. 59-+Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    We present a liquid chromatography-mass spectrometry (LC-MSMS)-based method permitting unbiased (gene prediction-independent) genome-wide discovery of protein-coding loci in higher eukaryotes. Using high-resolution isoelectric focusing (HiRIEF) at the peptide level in the 3.7-5.0 pH range and accurate peptide isoelectric point (pI) prediction, we probed the six-reading-frame translation of the human and mouse genomes and identified 98 and 52 previously undiscovered protein-coding loci, respectively. The method also enabled deep proteome coverage, identifying 13,078 human and 10,637 mouse proteins.

  • 114.
    Brehwens, Karl
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    In vitro and in vivo aspects of intrinsic radiosensitivity2014Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    This thesis focuses on how physical and biological factors influence the outcome of exposures to γ/X-rays. That the dose rate changes during real life exposure scenarios is well-known, but radiobiological data from exposures performed at increasing or decreasing dose rates is lacking. In paper I, it was found that an exposure where the dose rate decreases exponentially induces significantly higher levels of micronuclei in TK6 cells than exposures at an increasing or constant dose rate. Paper II describes the construction and validation of novel exposure equipment used to further study this “decreasing dose rate effect”, which is described in paper III. In paper I we also observed a radioprotective effect when cells were exposed on ice. This “temperature effect” (TE) has been known for decades but it is still not fully understood how hypothermia acts in a radioprotective manner. This was investigated in paper IV, where a multiparametric approach was used to investigate the underlying mechanisms. In paper V the aim was to investigate the role of biomarkers and clinical parameters as possible risk factors for late adverse effects to radiotherapy (RT). This was studied in a rare cohort of head-and-neck cancer patients that developed mandibular osteoradionecrosis (ORN) as a severe late adverse effect of RT. Biomarker measurements and clinical factors were then subjected to multivariate analysis in order to identify ORN risk factors. The results suggest that the patient’s oxidative stress response is an important factor in ORN pathogenesis, and support the current view that patient-related factors constitute the largest source of variation seen in the frequency of late adverse effects to RT.

    In summary, this thesis provides new and important insights into the roles of biological and physical factors in determining the consequences of γ/X-ray exposures.

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  • 115.
    Brehwens, Karl
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Bajinskis, Ainars
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. University of Latvia, Latvia.
    Haghdoost, Siamak
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut. Jan Kochanowski University, Poland.
    Micronucleus frequencies and clonogenic cell survival in TK6 cells exposed to changing dose rates under controlled temperature conditions2014Ingår i: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 90, nr 3, s. 241-247Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Purpose: In most exposure scenarios the dose rate of exposure is not constant. Despite this, very little information exists on the possible biological effects of exposing cells to radiation under the conditions of a changing dose rate. The current study highlights interesting effects following exposure under these conditions.

    Materials and methods: We constructed a new exposure facility that allows exposing cells inside an incubator and used it to irradiate human lymphoblastoid TK6 cells both after a moderate (0.48 Gy) and a high (1.1 Gy) dose, where the change in dose rate was, respectively, ≈ 17-fold change (2.2 - 37 mGy/min) and ≈ 39-fold (2.7 - 106 mGy/min). Clonogenic survival and micronuclei (MN) induction were the chosen endpoints.

    Results: The obtained results confirm the outcome of our first study that TK6 cells exposed to a decreasing dose rate express more MN than cells exposed to an increasing or constant dose rate. The effect was not seen after the moderate dose of 0.48 Gy or detectable at the level of clonogenic cell survival.

    Conclusions: We speculate that the high level of MN is probably related to a delayed elimination of damaged cells by interphase death, as opposed to mechanisms relating to DNA damage and repair.

  • 116.
    Brindefalk, Björn
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Botaniska institutionen.
    Dessailly, Benoit H.
    Yeats, Corin
    Orengo, Christine
    Werner, Finn
    Poole, Anthony M.
    Evolutionary history of the TBP-domain superfamily2013Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 41, nr 5, s. 2832-2845Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The TATA binding protein (TBP) is an essential transcription initiation factor in Archaea and Eucarya. Bacteria lack TBP, and instead use sigma factors for transcription initiation. TBP has a symmetric structure comprising two repeated TBP domains. Using sequence, structural and phylogenetic analyses, we examine the distribution and evolutionary history of the TBP domain, a member of the helix-grip fold family. Our analyses reveal a broader distribution than for TBP, with TBP-domains being present across all three domains of life. In contrast to TBP, all other characterized examples of the TBP domain are present as single copies, primarily within multidomain proteins. The presence of the TBP domain in the ubiquitous DNA glycosylases suggests that this fold traces back to the ancestor of all three domains of life. The TBP domain is also found in RNase HIII, and phylogenetic analyses show that RNase HIII has evolved from bacterial RNase HII via TBP-domain fusion. Finally, our comparative genomic screens confirm and extend earlier reports of proteins consisting of a single TBP domain among some Archaea. These monopartite TBP-domain proteins suggest that this domain is functional in its own right, and that the TBP domain could have first evolved as an independent protein, which was later recruited in different contexts.

  • 117.
    Bristulf, Jesper
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Interleukin-1 receptors and their ligands1994Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 118.
    Bromstrup, Torben
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Howard, Rebecca J.
    Trudell, James R.
    Harris, R. Adron
    Lindahl, Erik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Inhibition versus Potentiation of Ligand-Gated Ion Channels Can Be Altered by a Single Mutation that Moves Ligands between Intra- and Intersubunit Sites2013Ingår i: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, nr 8, s. 1307-1316Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Pentameric ligand-gated ion channels (pLGICs) are similar in structure but either inhibited or potentiated by alcohols and anesthetics. This dual modulation has previously not been understood, but the determination of X-ray structures of prokaryotic GLIC provides an ideal model system. Here, we show that a single-site mutation at the F14' site in the GLIC transmembrane domain turns desflurane and chloroform from inhibitors to potentiators, and that this is explained by competing allosteric sites. The F14'A mutation opens an intersubunit site lined by N239 (15'), 1240 (16'), and Y263. Free energy calculations confirm this site is the preferred binding location for desflurane and chloroform in GLIC F14'A. In contrast, both anesthetics prefer an intrasubunit site in wild-type GLIC. Modulation is therefore the net effect of competitive binding between the intersubunit potentiating site and an intrasubunit inhibitory site. This provides direct evidence for a dual-site model of allosteric regulation of pLGICs.

  • 119.
    Brostedt, Erica
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Electron transport to nitrogenase in the photosynthetic bacterium Rhodospirillum rubrum1994Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 120. Brunnström, Åsa
    et al.
    Tryselius, Ylva
    Feltenmark, Stina
    Andersson, Erik
    Leksell, Helene
    James, Anna
    Mannervik, Bengt
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Dahlén, Barbro
    Claesson, Hans-Erik
    On the biosynthesis of 15-HETE and eoxin C-4 by human airway epithelial cells2015Ingår i: Prostaglandins & other lipid mediators, ISSN 1098-8823, E-ISSN 2212-196X, Vol. 121, s. 83-90Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Several lines of evidence indicate that 15-lipoxygenase type 1 (15-LO-1) plays a pathophysiological role in asthma. The aim for this study was to investigate the 15-LO-1 expression and activity in primary human airway epithelial cells cultivated on micro-porous filters at air liquid interface. Incubation of human airway epithelial cells with arachidonic acid led to the formation of 15(S)-hydroxy-eicosatetraenoic acid (15-HETE) and exposing the cells to bacteria or physical injury markedly increased their production of 15-HETE. The cells were also found to convert arachidonic acid to eoxin C-4 (EXC4). Subcellular fractionation revealed that the conversion of EXA(4) to EXC4 was catalyzed by a soluble glutathione transferase (GST). The GST P1-1 enzyme was found to possess the highest activity of the investigated soluble GSTs. Following IL-4 treatment of airway epithelial cells, microarray analysis confirmed high expression of 15-LO-1 and GST P1-1, and immunohistochemical staining of bronchial biopsies revealed co-localization of 15-LO-1 and GST P1-1 in airway epithelial cells. These results indicate that respiratory infection and cell injury may activate the 15-LO pathway in airway epithelial cells. Furthermore, we also demonstrate that airway epithelial cells have the capacity to produce EXC4.

  • 121.
    Brzezinski, Peter
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Öjemyr, Linda Näsvik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Ädelroth, Pia
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Intermediates generated during the reaction of reduced Rhodobacter sphaeroides cytochrome c oxidase with dioxygen2013Ingår i: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1827, nr 7, s. 843-847Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    Cytochrome oxidase is one of the functionally most intriguing redox-driven proton pumps. During the last decade our increased understanding of the system has greatly benefited from theoretical calculations and modeling in the framework of three-dimensional structures of cytochrome c oxidases from different species. Because these studies are based on results from experiments, it is important that any ambiguities in the conclusions extracted from these experiments are discussed and elucidated. In a recent study Szundi et al. (Szundi et al. Biochemistry 2012, 51, 9302) investigated the reaction of the reduced Rhodobacter sphaeroides cytochrome c oxidase with O-2 and arrived at conclusions different from those derived from earlier investigations. In this short communication we compare these very recent data to those obtained from earlier studies and discuss the origin of the differences.

  • 122. Brzozowska, Kinga
    et al.
    Pinkawa, Michael
    Eble, Michael J.
    Muller, Wolfgang-Ullrich
    Wojcik, Andrzej
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Kriehuber, Ralf
    Schmitz, Sabine
    In vivo versus in vitro individual radiosensitivity analysed in healthy donors and in prostate cancer patients with and without severe side effects after radiotherapy2012Ingår i: International Journal of Radiation Biology, ISSN 0955-3002, E-ISSN 1362-3095, Vol. 88, nr 5, s. 405-413Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background : A high cellular radiosensitivity may be connected with a risk for development of severe side effects after radiotherapy and indicate cancer susceptibility. Hence, a fast and robust in vitro test is desirable to identify radiosensitive individuals. Materials and methods : The study included 25 prostate cancer patients with severe side effects (S) and 25 patients without severe side effects (0) after radiotherapy as well as 23 male healthy age-matched donors. Blood samples were exposed to 0.5 Gy or 1 Gy of gamma-rays. The initial level of double-strand breaks (dsb) and repair kinetics measured by phosphorylation of histone H2A (gamma-H2AX-assay), apoptosis (Annexin V-assay) and the induction of chromatid aberrations after irradiation in the G2-phase of the cell cycle (G2-assay) were analysed. Results : A significant higher chromatid aberration yield was found in lymphocytes from prostate cancer patients when compared to healthy donors. We found no significant differences between patients S and patients 0. Conclusions : There is no obvious correlation between clinical and cellular radiosensitivity in lymphocytes of prostate cancer patients when all chosen in vitro assays are considered. Although 25% of the patients showed both severe side effects and increased radiation-induced chromosomal sensitivity, predictive value of G2-assay is doubtful.

  • 123.
    Brändén, Gisela
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Structure and Function of Cytochrome c Oxidase2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Aerobic organisms, ranging from bacteria to humans, obtain their energy through the process of respiration. Electrons from the breakdown of food are transported through the membrane-bound enzyme complexes of the respiratory chain, to the terminal acceptor molecule, oxygen. The energy released in the process is used to transport protons across the cell membrane, thereby establishing an electrochemical gradient that can be utilised e.g. to synthesise ATP. The last respiratory complex in mitochondria and many bacteria is cytochrome c oxidase (CcO). CcO catalyses the reduction of oxygen to water, and at the same time pumps protons from the more negative to the more positive side of the membrane. In spite of extended and intensive research efforts, the detailed mechanism by which the exergonic electron-transfer reactions are coupled to the endergonic proton pumping is still not understood.

    The focus of this thesis is the structure and function of CcO from the bacterium Rhodobacter sphaeroides. The structures of the wild-type enzyme and two mutant enzymes have been investigated, providing insights into structural changes taking place upon replacing a glutamate residue (E286) in one of the proton-transfer pathways. E286 is a key element in directing protons either to the catalytic site for the oxygen chemistry or towards the outside for pumping. Results from time-resolved optical absorption spectroscopy show that the pKa of E286 has to be close to 9.5 for the proton pump to function, and that the pKa can be modulated by mutations at a distance of ~25 Å from E286. From another study we conclude that the D-propionate of haem a3 is most likely the acceptor site for pumped protons. Finally, we have identified the structural element which conformational changes determine the reaction rate of each proton-pumping step.

    The results are summarised in a molecular model for proton pumping by CcO, in which deprotonation of E286 drives proton translocation across the membrane.

  • 124.
    Bäckman, Hans G
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Functional and structural studies of the Presequence protease, PreP2014Licentiatavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2. 

    We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP.

    In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP.

    We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+

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  • 125.
    Calado Botelho, Salomé
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Translocation of proteins into and across the bacterial and mitochondrial inner membranes2012Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Translocons are dynamic protein complexes with the ability to respond to specific signals and to transport polypeptides between two distinct environments. The Sec-type translocons are examples of such machineries that can interconvert between a pore forming conformation that translocates proteins across the membrane, and a channel-like conformation that integrates proteins into the membrane by lateral opening.

    This thesis aims to identify the signals encoded in the amino acid sequence of the translocating polypeptides that trigger the translocon to release defined segments into the membrane. The selected systems are the SecYEG translocon and the TIM23 complex responsible for inserting proteins into the bacterial and the mitochondrial inner membrane, respectively.

    These two translocons have been challenged in vivo with designed polypeptide segments and their insertion efficiency into the membrane was measured. This allowed identification of the sequence requirements that govern SecYEG- and TIM23-mediated membrane integration. For these two systems, “biological” hydrophobicity scales have been determined, giving the contributions of each of the 20 amino acids to the overall free energy of insertion of a transmembrane segment into the membrane.

    A closer analysis of the mitochondrial system has made it possible to additionally investigate the process of membrane dislocation mediated by the m-AAA protease. The threshold hydrophobicity required for a transmembrane segment to remain in the mitochondrial inner membrane after TIM23-mediated integration depends on whether the segment will be further acted upon by the m-AAA protease.

    Finally, an experimental approach is presented to distinguish between different protein sorting pathways at the level of the TIM23 complex, i.e., conservative sorting vs. stop-transfer pathways. The results suggest a connection between the metabolic state of the cell and the import of proteins into the mitochondria.

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  • 126.
    Calado Botelho, Salomé
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Tatsuta, Takashi
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Kim, Hyun
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Seoul National University, South Korea.
    Dislocation by the m-AAA Protease Increases the Threshold Hydrophobicity for Retention of Transmembrane Helices in the Inner Membrane of Yeast Mitochondria2013Ingår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, nr 7, s. 4792-4798Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Sorting of mitochondrial inner membrane proteins is a complex process in which translocons and proteases function in a concerted way. Many inner membrane proteins insert into the membrane via the TIM23 translocon, and some are then further acted upon by the mitochondrial m-AAA protease, a molecular motor capable of dislocating proteins from the inner membrane. This raises the possibility that the threshold hydrophobicity for the retention of transmembrane segments in the inner membrane is different depending on whether they belong to membrane proteins that are m-AAA protease substrates or not. Here, using model transmembrane segments engineered into m-AAA protease-dependent proteins, we show that the threshold hydrophobicity for membrane retention measured in yeast cells in the absence of a functional m-AAA protease is markedly lower than that measured in its presence. Whether a given hydrophobic segment in a mitochondrial inner membrane protein will ultimately form a transmembrane helix may therefore depend on whether or not it will be exposed to the pulling force exerted by the m-AAA protease during biogenesis.

  • 127.
    Cardoso-Palacios, Carlos
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Sylwan, Lina
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Mandali, Sridhar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    Frumerie, Clara
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Haggård-Ljungquist, Elisabeth
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för genetik, mikrobiologi och toxikologi.
    A structure-function analysis of P2 integraseManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Bacteriophage P2 integrase catalyzes site-specific recombination between the phage DNA and the host chromosome thereby promoting integration or excision of the phage genome. P2 integrase belongs to the large tyrosine family of integrases that shows little sequence identity besides some conserved boxes and patches in the catalytic domain. However, the overall structure of the tyrosine family of integrases seems to be similar. Phage integrases have the potential as tools for site-specific gene insertions into eukaryotic genomes provided that target sequences are available. To elucidate the possibility of evolving the P2 integrase to accept new targets, we have in this work initiated a structure-function analysis of the P2 integrase using two approaches based on a comparison of the predicted secondary structure of P2 integrase with that determined for the lambda integrase. First, we have made hybrids between P2 integrase and the related WΦ integrase that has a different host DNA target, to locate the region promoting specificity between the integrases. This, however, has not been possible, the N-terminal domains can be exchanged without losing biological activity and this will not affect the specificity. All other hybrids made were biological inactive. Next we have made an alanine scanning of the alpha helices believed to be involved in specific interactions with the target, and four amino acids have been identified as candidates for sequence-specific interactions with the core.

  • 128. Carstensen, Jacob
    et al.
    Andersen, Jesper H.
    Gustafsson, Bo G.
    Stockholms universitet, Naturvetenskapliga fakulteten, Stockholms universitets Östersjöcentrum, Baltic Nest Institute.
    Conley, Daniel J.
    Deoxygenation of the Baltic Sea during the last century2014Ingår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 111, nr 15, s. 5628-5633Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Deoxygenation is a global problem in coastal and open regions of the ocean, and has led to expanding areas of oxygen minimum zones and coastal hypoxia. The recent expansion of hypoxia in coastal ecosystems has been primarily attributed to global warming and enhanced nutrient input from land and atmosphere. The largest anthropogenically induced hypoxic area in the world is the Baltic Sea, where the relative importance of physical forcing versus eutrophication is still debated. We have analyzed water column oxygen and salinity profiles to reconstruct oxygen and stratification conditions over the last 115 y and compare the influence of both climate and anthropogenic forcing on hypoxia. We report a 10-fold increase of hypoxia in the Baltic Sea and show that this is primarily linked to increased inputs of nutrients from land, although increased respiration from higher temperatures during the last two decades has contributed to worsening oxygen conditions. Although shifts in climate and physical circulation are important factors modulating the extent of hypoxia, further nutrient reductions in the Baltic Sea will be necessary to reduce the ecosystems impacts of deoxygenation.

  • 129.
    Cassel, Marika
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Studies on the Conformation of Transmembrane Polypeptides in Membrane Proteins2005Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    The major aim of the studies that this thesis is based on has been to better define the topological determinants of the formation of so-called helical hairpins during membrane protein assembly in the ER membrane.

    The helical hairpin is a basic folding unit in membrane proteins. It is composed of two closely spaced transmembrane helices with a short connecting loop and it is believed to be inserted into the membrane as one compact unit. It is becoming increasingly clear that the helical hairpin is a very common structural element in membrane proteins and a detailed understanding of its properties is of central importance.

    We demonstrate that the efficiency of formation of helical hairpins depends both on the overall length of the hydrophobic segment, on the amino acids flanking the transmembrane segment, and on the identity of the central, potentially turn-forming residues. We also show that interhelical hydrogen bonds between pairs of Asn or Asp residues can induce helical hairpin formation.

    A detailed topology mapping is also reported for the Escherichia coli inner membrane chloride channel YadQ, a protein for which the X-ray structure is known. Our results provide a critical test of the reporter fusion approach and offer new insights into the YadQ folding pathway.

    In summary, the results present in this thesis have increased our understanding of the determinants of membrane protein topology and structure. Furthermore, the information obtained can be used to improve current models for predictions of membrane protein topology.

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  • 130.
    Castro, Víctor M.
    Stockholms universitet.
    Significance of glutathione transferases in the resistance of tumor cells to alkylating cytostatic drugs1991Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 131.
    Celorio-Mancera, Maria de la Paz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Sundmalm, Sara M.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Vogel, Heiko
    Rutishauser, Dorothea
    Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Ytterberg, A. Jimmy
    Zubarev, Roman A.
    Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Janz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Chemosensory proteins, major salivary factors in caterpillar mandibular glands2012Ingår i: Insect Biochemistry and Molecular Biology, ISSN 0965-1748, E-ISSN 1879-0240, Vol. 42, nr 10, s. 796-805Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Research in the field of insect-host plant interactions has indicated that constituents of insect saliva play an important role in digestion and affect host chemical defense responses. However, most efforts have focused on studying the composition and function of regurgitant or saliva produced in the labial glands. Acknowledging the need for understanding the role of the mandibular glands in herbivory, we sought to make a qualitative and semi-quantitative comparison of soluble luminal fractions between mandibular and labial glands of Vanessa gonerilla butterfly larvae. Amylase and lysozyme were inspected as possible major enzymatic activities in the mandibular glands aiding in pre-digestion and antimicrobial defense. Although detected, neither of these enzymatic activities was prominent in the luminal protein preparation of a particular type of gland. Proteins isolated from the glands were identified by mass spectrometry and by searching an EST-library database generated for four other nymphalid butterfly species, in addition to the public NCBI database. The identified proteins were also quantified from thedata using “Quanty”, an in-house program. The proteomic analysis detected chemosensory proteins as the most abundant luminal proteins in the mandibular glands. In comparison to these proteins, the relative amounts of amylase and lysozyme were much lower in both gland types. Therefore, we speculate that the primary role of the mandibular glands in Lepidopteran larvae is chemoreception which may include the detection of microorganisms on plant surfaces, host plant recognition and communication with conspecifics.

  • 132.
    Celorio-Mancera, Maria de la Paz
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Wheat, Christopher W.
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för populationsgenetik.
    Vogel, Heiko
    Söderlind, Lina
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Janz, Niklas
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Nylin, Sören
    Stockholms universitet, Naturvetenskapliga fakulteten, Zoologiska institutionen, Avdelningen för zoologisk ekologi.
    Mechanisms of macroevolution: polyphagous plasticity in butterfly larvae revealed by RNA-Seq2013Ingår i: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 22, nr 19, s. 4884-4895Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transcriptome studies of insect herbivory are still rare, yet studies in model systems have uncovered patterns of transcript regulation that appear to provide insights into how insect herbivores attain polyphagy, such as a general increase in expression breadth and regulation of ribosomal, digestion- and detoxification-related genes. We investigated the potential generality of these emerging patterns, in the Swedish comma, Polygonia c-album, which is a polyphagous, widely-distributed butterfly. Urtica dioica and Ribes uva-crispa are hosts of P. c-album, but Ribes represents a recent evolutionary shift onto a very divergent host. Utilizing the assembled transcriptome for read mapping, we assessed gene expression finding that caterpillar life-history (i.e. 2nd vs. 4th-instar regulation) had a limited influence on gene expression plasticity. In contrast, differential expression in response to host-plant identified genes encoding serine-type endopeptidases, membrane-associated proteins and transporters. Differential regulation of genes involved in nucleic acid binding was also observed suggesting that polyphagy involves large scale transcriptional changes. Additionally, transcripts coding for structural constituents of the cuticle were differentially expressed in caterpillars in response to their diet indicating that the insect cuticle may be a target for plant defence. Our results state that emerging patterns of transcript regulation from model species appear relevant in species when placed in an evolutionary context.

  • 133.
    Cerrato, Carmine Pasquale
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Cell-Penetrating Peptides for Mitochondrial Targeting2018Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Mitochondria have simply been known as the cell’s powerhouse for a long time, with its vital function of producing ATP. However, substantially more attention was directed towards these organelles once they were recognized to perform several essential functions having an impact in cell biology, pharmaceutics and medicine. Dysfunctions of these organelles have been linked to several diseases such as diabetes, cancer, neurodegenerative diseases and cardiovascular disorders. Mitochondrial medicine emerged once the relationship of reactive oxygen species and mutations of the mitochondrial DNA linked to diseases was shown, referred to as mitochondrial dysfunction. This has led to the need to deliver therapeutic molecules in their active form not only to the target cells but more importantly into the targeted organelles.

    In this thesis, cell-penetrating peptides (CPPs) used as mitochondrial drug delivery system and the pathways involved in the uptake mechanisms of a CPP are described. In particular, Paper I describes a novel cell-penetrating peptide targeting mitochondria with intrinsic antioxidant properties. Paper II expands upon this first finding and show that the same peptide can carry a glutathione analogue peptide with improved radical scavenging ability into cytoplasm and mitochondria. Paper III introduces mitochondrial targeting peptides for delivery of therapeutic biomolecules to modify mitochondrial gene expression. In Paper IV, the uptake mechanisms of the CPP delivery strategy has been investigated to gain a better understanding of the used transfection system.

    Overall, this thesis summarizes our current effort regarding cell-penetrating peptides delivery system to target mitochondria and the progress made towards a potential gene therapy. It contributes to the field of CPPs and drug delivery with a set of peptides with radical scavenging ability, a strategy to deliver oligonucleotides to mitochondria as proof-of-concept for mitochondrial gene therapy, and to help understanding the pathways involved in CPPs uptake.

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  • 134.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Kivijärvi, Tove
    Tozzi, Roberta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Peptides targeting mitochondria for efficient delivery of therapeutic biomoleculesManuskript (preprint) (Övrigt vetenskapligt)
  • 135. Cerrato, Carmine Pasquale
    et al.
    Kivijärvi, Tove
    Tozzi, Roberta
    Lehto, Tõnis
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Gestin, Maxime
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Intracellular Delivery of Therapeutic Antisense Oligonucleotides Targeting mRNACoding Mitochondrial Proteins by Cell-Penetrating PeptidesManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Cell-penetrating peptides are a promising therapeutic strategy for a wide variety of degenerative diseases, ageing, and cancer. Among the multitude of cellpenetrating peptides, PepFect14 has been preferentially used in our laboratory for oligonucleotide delivery into cells and in vivo mouse models. However, this activity has mainly been reported towards cytoplasm and nuclei, while the mentioned disorders have been linked to mitochondrial defects. Here, we report a library generated from a combinatorial covalent fusion of a mitochondrial-penetrating peptide, mtCPP1, and PepFect14 in order to deliver therapeutic biomolecules to influence mitochondrial protein expression. The non-covalent complexation of these peptides with oligonucleotides resulted in nanocomplexes affecting biological functions in the cytoplasm and on mitochondria. This delivery system proved to efficiently target mitochondrial genes, providing a framework for the development of mitochondrial peptide-based oligonucleotide technologies with the potential to be used as a treatment for patients with mitochondrial disorders.

  • 136.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Künnapuu, Kadri
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi. University of Tartu, Estonia.
    Cell-penetrating peptides with intracellular organelle targeting2017Ingår i: Expert Opinion on Drug Delivery, ISSN 1742-5247, E-ISSN 1744-7593, Vol. 14, nr 2, s. 245-255Artikel, forskningsöversikt (Refereegranskat)
    Abstract [en]

    INTRODUCTION: One of the major limiting steps in order to have an effective drug is the passage through one or more cell membranes to reach its site of action. To reach the action-site, the specific macromolecules are required to be delivered specifically to the cell compartment/organelle in their (pre)active form.

    AREAS COVERED: In this review, we will discuss cell-penetrating peptides (CPPs) developed in the last decade to transport small RNA/DNA, plasmids, antibodies, and nanoparticles into specific sites of the cell. The article describes CPPs in complex with cargo molecules that target specific intracellular organelles and their potential for pharmacological or clinical use.

    EXPERT OPINION: Organelle targeting is the ultimate goal to ensure selective delivery to the site of action in the cells. 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 genomic diseases as well as infections, cancer, neurodegenerative and hereditary diseases. They have proven to be successful in delivering various therapeutic agents into cells however, further in vivo experiments and clinical trials are required to demonstrate the efficacy of this technology.

  • 137.
    Cerrato, Carmine Pasquale
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. University of Tartu, Estonia.
    Cell-Penetrating Peptides Targeting Mitochondria2018Ingår i: Mitochondrial Biology and Experimental Therapeutics / [ed] Paulo J. Oliveira, Cham: Springer, 2018, s. 593-611Kapitel i bok, del av antologi (Refereegranskat)
    Abstract [en]

    Mitochondria are key organelles with essential functions and fundamental roles in cell death and survival signaling. Consequently, they are involved in a wide range of diseases with a great diversity of clinical appearance, which makes them attractive as target for drugs to treat metabolic and degenerative diseases and cancer. Efficient methods for specific intracellular delivery of exogenous compounds, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles, would be beneficial for research and patients. A sustained effort in the last 20 years has been done to exploit cell-penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro  and in vivo  because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the mechanisms by which CPPs can function, the use of this alternative as well as strategies used to target mitochondria and the implications for drug delivery.

  • 138.
    Cerrato, Carmine
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Veiman, Kadi-Liis
    Laboratory of Molecular Biotechnology, Institute of Technology, Tartu University, Tartu, Estonia.
    Langel, Ülo
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för neurokemi.
    Advances in peptide delivery2015Ingår i: Advances in the Discovery and Development of Peptide Therapeutics, Future Science Group , 2015, s. 160-171Kapitel i bok, del av antologi (Refereegranskat)
  • 139. Cervin, Nicholas T.
    et al.
    Andersson, Linnea
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Ng, Jovice Boon Sing
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Olin, Pontus
    Bergström, Lennart
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för material- och miljökemi (MMK).
    Wågberg, Lars
    Lightweight and Strong Cellulose Materials Made from Aqueous Foams Stabilized by Nanofibrillated Cellulose2013Ingår i: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 14, nr 2, s. 503-511Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    A lightweight and strong porous cellulose material has been prepared by drying aqueous foams stabilized with surface-modified nanofibrillated cellulose (NFC). This material differs from other dry, particle stabilized foams in that renewable cellulose is used as stabilizing particles. Confocal microscopy and high speed video imaging show that the octylamine-coated, rod-shaped NFC nanoparticles residing at the air-liquid interface prevent the air bubbles from collapsing or coalescing. Stable wet foams can be achieved at solids content around 1% by weight. Careful removal of the water results in a cellulose-based material with a porosity of 98% and a density of 30 mg cm(-3). These porous cellulose materials have a higher Young's modulus than porous cellulose materials made from freeze-drying, at comparable densities, and have a compressive energy absorption of 56 kJ m(-3) at 80% strain. Measurement with the aid of an autoporosimeter revealed that most pores are in the range of 300 to 500 mu m.

  • 140.
    Chammiran, Daniel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Veno, Morten T.
    Ekdahl, Ylva
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    Kjems, Jörgen
    Öhman, Marie
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylärbiologi och funktionsgenomik.
    A distant cis acting intronic element induces site-selective RNA editing2012Ingår i: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 40, nr 19, s. 9876-9886Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome.

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  • 141. Chen, Jue
    et al.
    Teixeira, Pedro Filipe
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Glaser, Elzbieta
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Levine, Rodney L.
    Mechanism of oxidative inactivation of human presequence protease by hydrogen peroxide2014Ingår i: Free Radical Biology & Medicine, ISSN 0891-5849, E-ISSN 1873-4596, Vol. 77, s. 57-63Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The mitochondrial presequence protease (PreP) is a member of the pitrilysin class of metalloproteases. It degrades the mitochondrial targeting presequences of mitochondria-localized proteins as well as unstructured peptides such as amyloid-beta peptide. The specific activity of PreP is reduced in Alzheimer patients and animal models of Alzheimer disease. The loss of activity can be mimicked in vitro by exposure to oxidizing conditions, and indirect evidence suggested that inactivation was due to methionine oxidation. We performed peptide mapping analyses to elucidate the mechanism of inactivation. None of the 24 methionine residues in recombinant human PreP was oxidized. We present evidence that inactivation is due to oxidation of cysteine residues and consequent oligomerization through intermolecular disulfide bonds. The most susceptible cysteine residues to oxidation are Cys34, Cys112, and Cys119. Most, but not all, of the activity loss is restored by the reducing agent dithiothreitol. These findings elucidate a redox mechanism for regulation of PreP and also provide a rational basis for therapeutic intervention in conditions characterized by excessive oxidation of PreP.

  • 142.
    Chiruvella, Kishore K.
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Rajaei, Naghmeh
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Jonna, Venkateswara Rao
    Hofer, Anders
    Åström, Stefan U.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för molekylär biovetenskap, Wenner-Grens institut.
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, artikel-id 21671Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Kluyveromyces lactis hAT-transposase 1 (Kat1) generates hairpin-capped DNA double strand breaks leading to MAT-switching (MATa to MAT alpha). Using purified Kat1, we demonstrate the importance of terminal inverted repeats and subterminal repeats for its endonuclease activity. Kat1 promoted joining of the transposon end into a target DNA molecule in vitro, a biochemical feature that ties Kat1 to transposases. Gas-phase Electrophoretic Mobility Macromolecule analysis revealed that Kat1 can form hexamers when complexed with DNA. Kat1 point mutants were generated in conserved positions to explore structure-function relationships. Mutants of predicted catalytic residues abolished both DNA cleavage and strand-transfer. Interestingly, W576A predicted to be impaired for hairpin formation, was active for DNA cleavage and supported wild type levels of mating-type switching. In contrast, the conserved CXXH motif was critical for hairpin formation because Kat1 C402A/H405A completely blocked hairpinning and switching, but still generated nicks in the DNA. Mutations in the BED zinc-finger domain (C130A/C133A) resulted in an unspecific nuclease activity, presumably due to nonspecific DNA interaction. Kat1 mutants that were defective for cleavage in vitro were also defective for mating-type switching. Collectively, this study reveals Kat1 sharing extensive biochemical similarities with cut and paste transposons despite being domesticated and evolutionary diverged from active transposons.

  • 143.
    Ciftci, Sibel
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Padlock Probe-Based Nucleic Acid Amplification Tests: Point-of-care Diagnostics of Infectious Diseases2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Recent advancements in molecular biology and biotechnology have pushed the field of molecular diagnostics much further to benefit the society towards smart access for rapid and simplified health care. In this context, point-of-care (PoC) technologies that bring the inventions in diagnostics closer to bedside settings draw attention. This becomes all the more relevant in the case of infectious diseases which pose the major burden, in terms of mortality and economic loss, especially for third world developing countries with resource-limited settings (RLS). Moreover, emerging and re-emerging viruses, known for their rapid mutation rates, demand huge attention in terms of timely diagnosis and the need for effective treatments. Hence, appropriate and accurate tests to detect the pathogens with enhanced sensitivity and specificity would be needed to bridge the gap between bioanalytics and clinics.

    This research work is an attempt to combine the tools and techniques required for the development of such efficient PoC technologies to combat infectious diseases. Among available nucleic acid-based amplification tests, padlock probing and isothermal rolling circle amplification are used to benefit from the advantages they offer for diagnostic applications, in terms of specificity, multiplexability, single molecule detection, high throughput, compatibility with various read-out platforms and inexpensive digital quantification.

    In the first paper, simultaneous detection of RNA and DNA forms of adenovirus is shown to study the spatio-temporal expression patterns in both lytic and persistent infections. In situ quantification of viral DNA as well as transcripts with single cell resolution has been achieved. In the second paper, novel probe design strategy has been presented for the development of molecular assays to detect hypervariable RNA viruses. This approach becomes helpful in targeting rapidly evolving viruses by using mutation-tolerant probes for RCA. Third paper demonstrates simple RCA for rapid detection of Ebola virus in clinical samples, followed by a multiplexed detection with other re-emerging tropical viruses, namely Zika and Dengue. This study also includes a simple easy-to-operate pump-free membrane enrichment read-out, combined together with microscopy for digital quantification of the products. In the fourth paper, near point-of-care glucose sensor-based RCP detection has been proposed for Ebola virus detection. All these attempts clearly bring RCA closer to PoC settings for molecular diagnostics of virus infections.

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  • 144.
    Ciftci, Sibel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Abdurahman, Samir
    Appelberg, Sofia
    Mirazimi, Ali
    Madaboosi, Narayanan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Multiplexed rolling circle amplification detection of Ebola, Zika and Dengue towards point-of-care diagnosticsManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Emerging tropical viruses have caused serious outbreaks during the recent years, such as Ebola virus (EBOV) in 2014 and the most recent 2018-19 outbreak in Congo. Immediate diagnostic attention is demanded, and most importantly at the point-of-care in resource-limited settings. The performance and the operational parameters of conventional EBOV testing are limited by either their sensitivity, specificity, or both, and often do not cover other tropical disease viruses. We present a padlock probe (PLP)-based rolling circle amplification (RCA) method for the detection of EBOV from cell culture isolates as well as clinical samples obtained from patients of West Africa outbreak. For this, a set of PLPs, separately targeting the vRNA and cRNA of all the seven genes of EBOV, were used in the RCA and validated on virus isolates from cell culture. The assay was then translated for testing clinical samples, and simultaneous duplex detection of both EBOV vRNA and cRNA was demonstrated. For increased sensitivity, the RCA products were enriched on a simple and pump-free microfluidic chip. As PLPs and RCA are inherently mulitplexable, we demonstrate the extension of the probe panel to the simultaneous detection of the tropical viruses Ebola, Zika and Dengue. The simple, rapid, specific and multiplexable isothermal assay developed for tropical virus detection suits the point-of-care needs, bringing RCA a step closer to bedside diagnostics.

  • 145.
    Ciftci, Sibel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Hernández-Neuta, Iván
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Hakhverdyan, Mikhayil
    Bálint, Ádám
    Herthnek, David
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Madaboosi, Narayanan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    A novel mutation tolerant padlock probe design for multiplexed detection of hypervariable RNA viruses2019Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, artikel-id 2872Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The establishment of a robust detection platform for RNA viruses still remains a challenge in molecular diagnostics due to their high mutation rates. Newcastle disease virus (NDV) is one such RNA avian virus with a hypervariable genome and multiple genotypes. Classical approaches like virus isolation, serology, immunoassays and RT-PCR are cumbersome, and limited in terms of specificity and sensitivity. Padlock probes (PLPs) are known for allowing the detection of multiple nucleic acid targets with high specificity, and in combination with Rolling circle amplification (RCA) have permitted the development of versatile pathogen detection assays. In this work, we aimed to detect hypervariable viruses by developing a novel PLP design strategy capable of tolerating mutations while preserving high specificity by targeting several moderately conserved regions and using degenerate bases. For this, we designed nine padlock probes based on the alignment of 335 sequences covering both Class I and II NDV. Our PLP design showed high coverage and specificity for the detection of eight out of ten reported genotypes of Class II NDV field isolated strains, yielding a detection limit of less than ten copies of viral RNA. Further taking advantage of the multiplex capability of PLPs, we successfully extended the assay for the simultaneous detection of three poultry RNA viruses (NDV, IBV and AIV) and combined it with a paper based microfluidic enrichment read-out for digital quantification. In summary, our novel PLP design addresses the current issue of tolerating mutations of highly emerging virus strains with high sensitivity and specificity.

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  • 146.
    Ciftci, Sibel
    et al.
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Neumann, Felix
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Paulraj, Thomas
    Crespo, Gaston
    Madaboosi, Narayanan
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    Nilsson, Mats
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik. Stockholms universitet, Science for Life Laboratory (SciLifeLab).
    The sweet detection of rolling circle amplification: Glucose-based electrochemical detection of virus nucleic acidManuskript (preprint) (Övrigt vetenskapligt)
    Abstract [en]

    Infectious diseases remain a constant threat on a global scale by recurring pandemics. Rapid and portable diagnostics hold the promise to tackle the spreading of diseases and decentralizing healthcare to point-of-care needs. Ebola, a hypervariable RNA virus causing fatalities of up to 90% for recent outbreaks in Africa, demands immediate attention for bedside diagnostics. Nucleic acid amplification technology (NAAT) has proven to be a powerful tool for the control of outbreak with high sensitivity and specificity. However, NAAT is mostly based on amplification methods that require specialized instrumentation and trained personnel, such as PCR with sophisticated detectors. Here, we present an isothermal padlock probe-based assay for the detection of pathogens coupled with a glucose oxidase (GOx)-based electrochemical approach as the read-out. The assay design is based on rolling circle amplification (RCA) upon magnetic beads, connecting the RCA products (RCPs) via streptavidin-biotin bridges to GOx needed for the electrochemical measurement with externally provided glucose. The RCPs forming on the surface of beads are imaged using scanning electron microscopy, and the presence of the GOx to the RCP complex is confirmed using atomic force microscopy. Parameters such as the choice of buffers, concentrations of glucose and GOx and measurement time were optimized, as well as the mode of addition of glucose was tested. 125 μg/mL of GOx with 5 mM glucose using PBS as washing buffer, monitored for 15 min were chosen as the optimized conditions. The effect of temperature was tested and found to be critical at 37 °C for enhanced performance of the sensor. Finally, we evaluate the analytical performance of our sensor system by using cell culture isolate and clinical samples of Ebola virus. The study provides a proof-of-concept of simple and portable molecular diagnostics for emerging pathogens, beneficial especially for resource-limited settings. 

  • 147.
    Clinchy, Birgitta
    Stockholms universitet, Naturvetenskapliga fakulteten.
    Cytokine regulation of motile responses in B lymphocytes1994Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
  • 148. Coelho, Miguel
    et al.
    Lade, Steven J.
    Stockholms universitet, Naturvetenskapliga fakulteten, Stockholm Resilience Centre. Max Planck Institute for the Physics of Complex Systems, Germany.
    Alberti, Simon
    Gross, Thilo
    Tolic, Iva M.
    Fusion of Protein Aggregates Facilitates Asymmetric Damage Segregation2014Ingår i: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 12, nr 6, s. e1001886-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Asymmetric segregation of damaged proteins at cell division generates a cell that retains damage and a clean cell that supports population survival. In cells that divide asymmetrically, such as Saccharomyces cerevisiae, segregation of damaged proteins is achieved by retention and active transport. We have previously shown that in the symmetrically dividing Schizosaccharomyces pombe there is a transition between symmetric and asymmetric segregation of damaged proteins. Yet how this transition and generation of damage-free cells are achieved remained unknown. Here, by combining in vivo imaging of Hsp104-associated aggregates, a form of damage, with mathematical modeling, we find that fusion of protein aggregates facilitates asymmetric segregation. Our model predicts that, after stress, the increased number of aggregates fuse into a single large unit, which is inherited asymmetrically by one daughter cell, whereas the other one is born clean. We experimentally confirmed that fusion increases segregation asymmetry, for a range of stresses, and identified Hsp16 as a fusion factor. Our work shows that fusion of protein aggregates promotes the formation of damage-free cells. Fusion of cellular factors may represent a general mechanism for their asymmetric segregation at division.

  • 149. Collins, Ruairi
    et al.
    Johansson, Ann-Louise
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Karlberg, Tobias
    Markova, Natalia
    van den Berg, Susanne
    Olesen, Kenneth
    Hammarstrom, Martin
    Flores, Alex
    Schuler, Herwig
    Schiavone, Lovisa Holmberg
    Brzezinski, Peter
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Arner, Elias S. J.
    Högbom, Martin
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Biochemical Discrimination between Selenium and Sulfur 1: A Single Residue Provides Selenium Specificity to Human Selenocysteine Lyase2012Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, nr 1, s. e30581-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

  • 150. Contreras, F.-Xabier
    et al.
    Ernst, Andreas M.
    Haberkant, Per
    Björkholm, Patrik
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Lindahl, Erik
    Gönen, Basak
    Tischer, Christian
    Elofsson, Arne
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    von Heijne, Gunnar
    Stockholms universitet, Naturvetenskapliga fakulteten, Institutionen för biokemi och biofysik.
    Thiele, Christoph
    Pepperkok, Rainer
    Wieland, Felix
    Brügger, Britta
    Molecular recognition of a single sphingolipid species by a protein’s transmembrane domain2012Ingår i: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 481, nr 7382, s. 525-529Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.

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