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  • 1. 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.

  • 2. Ali, Raja Hashim
    et al.
    Muhammad, Sayyed Auwn
    Khan, Mehmood Alam
    Arvestad, Lars
    Stockholm University, Faculty of Science, Numerical Analysis and Computer Science (NADA). Stockholm University, Science for Life Laboratory (SciLifeLab). Swedish e-Science Research Center, Sweden .
    Quantitative synteny scoring improves homology inference and partitioning of gene families2013In: BMC Bioinformatics, E-ISSN 1471-2105, Vol. 14, no Suppl,15, p. S12-Article in journal (Refereed)
    Abstract [en]

    Background

    Clustering sequences into families has long been an important step in characterization of genes and proteins. There are many algorithms developed for this purpose, most of which are based on either direct similarity between gene pairs or some sort of network structure, where weights on edges of constructed graphs are based on similarity. However, conserved synteny is an important signal that can help distinguish homology and it has not been utilized to its fullest potential.

    Results

    Here, we present GenFamClust, a pipeline that combines the network properties of sequence similarity and synteny to assess homology relationship and merge known homologs into groups of gene families. GenFamClust identifies homologs in a more informed and accurate manner as compared to similarity based approaches. We tested our method against the Neighborhood Correlation method on two diverse datasets consisting of fully sequenced genomes of eukaryotes and synthetic data.

    Conclusions

    The results obtained from both datasets confirm that synteny helps determine homology and GenFamClust improves on Neighborhood Correlation method. The accuracy as well as the definition of synteny scores is the most valuable contribution of GenFamClust.

  • 3.
    Alikhani, Nyosha
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The Mitochondrial Peptidasome, PreP, relation to Alzheimer Disease2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Amyloid-β (Aβ) is the toxic peptide implicated in the pathogenesis of Alzheimer Disease (AD). Accumulation of Aβ has been shown in brain mitochondria from AD patients and AD mice models. The occurrence of Aβ in the mitochondrial matrix leads to free radical generation and apoptosis in neurons.

    In our studies, Aβ was found in brain mitochondria of living patients with plaque pathology. Extracellular Aβ was taken up by neuroblastoma cells and was found in the mitochondria. Moreover, we showed that Aβ40 and Aβ42 are transported into mitochondria via the Translocase of the Outer Membrane, the TOM machinery.

    We have identified the mitochondrial Aβ-degrading protease hPreP, in human brain mitochondrial matrix. PreP is a metalloprotease, originally identified as presequence protease, but was also shown to degrade other unstructured peptides including Aβ. Immunoinactivation of PreP in human brain mitochondria revealed PreP to be the protease responsible for Aβ degradation in mitochondria.

    Also, we have investigated if genetic variation in the gene encoding hPreP is associated with AD by genotyping 19 single nucleotide polymorphisms (SNPs) in the Swedish population. The study did not show a genetic association between any of the genotyped SNPs and the risk to AD. However, the biochemical analysis of four SNPs selected on the basis of their location within a structural homology model of hPreP revealed a decreased activity compared to wildtype.

    Interestingly, the activity of PreP in human brain mitochondrial matrix in AD individuals was significantly lower compared to non-dement aged-matched controls. These findings were also confirmed in brain mitochondrial matrix of AD mouse models. These results suggest that a decreased PreP activity may contribute to Aβ aggregation and accumulation inside mitochondria leading to neuronal death in AD. In summary, our findings show that the degradation of Aβ by hPreP may be of importance in the pathology of AD.

  • 4.
    Almamoun, Radwa
    et al.
    Stockholm University, Faculty of Science, Department of Environmental Science. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Pierozan, Paula
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Environmental Science.
    Karlsson, Oskar
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Environmental Science.
    Mechanistic screening of reproductive toxicity in a novel 3D testicular co-culture model shows significant impairments following exposure to low-dibutyl phthalate concentrations2024In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738Article in journal (Refereed)
    Abstract [en]

    To improve the mechanistic screening of reproductive toxicants in  chemical-risk assessment and drug development, we have developed a three-dimensional (3D) heterogenous testicular co-culture model from neonatal mice. Di-n-butyl phthalate (DBP), an environmental contaminant that can affect reproductive health negatively, was used as a model compound to illustrate the utility of the in vitro model. The cells were treated with DBP (1 nM to 100 µM) for 7 days. Automated high-content imaging confirmed the presence of cell-specific markers of Leydig cells (CYP11A1 +), Sertoli cells (SOX9 +), and germ cells (DAZL +). Steroidogenic activity of Leydig cells was demonstrated by analyzing testosterone levels in the culture medium. DBP induced a concentration-dependent reduction in testosterone levels and decreased the number of Leydig cells compared to vehicle control. The levels of steroidogenic regulator StAR and the steroidogenic enzyme CYP11A1 were decreased already at the lowest DBP concentration (1 nM), demonstrating upstream effects in the testosterone biosynthesis pathway. Furthermore, exposure to 10 nM DBP decreased the levels of the germ cell-specific RNA binding protein DAZL, central for the spermatogenesis. The 3D model also captured the development of the Sertoli cell junction proteins, N-cadherin and Zonula occludens protein 1 (ZO-1), critical for the blood–testis barrier. However, DBP exposure did not significantly alter the cadherin and ZO-1 levels. Altogether, this 3D in vitro system models testicular cellular signaling and function, making it a powerful tool for mechanistic screening of developmental testicular toxicity. This can open a new avenue for high throughput screening of chemically-induced reproductive toxicity during sensitive developmental phases.

  • 5. An, Rong
    et al.
    Wu, Nanhua
    Gao, Qingwei
    Dong, Yihui
    Laaksonen, Aatto
    Stockholm University, Science for Life Laboratory (SciLifeLab). Stockholm University, Faculty of Science, Department of Materials and Environmental Chemistry (MMK), Physical Chemistry. Luleå University of Technology, Sweden; ‘‘Petru Poni” Institute of Macromolecular Chemistry, Romania; Nanjing Tech University, China.
    Shah, Faiz Ullah
    Ji, Xiaoyan
    Fuchs, Harald
    Integrative studies of ionic liquid interface layers: bridging experiments, theoretical models and simulations2024In: Nanoscale Horizons, ISSN 2055-6764, E-ISSN 2055-6756Article, review/survey (Refereed)
    Abstract [en]

    Ionic liquids (ILs) are a class of salts existing in the liquid state below 100 degrees C, possessing low volatility, high thermal stability as well as many highly attractive solvent and electrochemical capabilities, etc., making them highly tunable for a great variety of applications, such as lubricants, electrolytes, and soft functional materials. In many applications, ILs are first either physi- or chemisorbed on a solid surface to successively create more functional materials. The functions of ILs at solid surfaces can differ considerably from those of bulk ILs, mainly due to distinct interfacial layers with tunable structures resulting in new ionic liquid interface layer properties and enhanced performance. Due to an almost infinite number of possible combinations among the cations and anions to form ILs, the diversity of various solid surfaces, as well as different external conditions and stimuli, a detailed molecular-level understanding of their structure-property relationship is of utmost significance for a judicious design of IL-solid interfaces with appropriate properties for task-specific applications. Many experimental techniques, such as atomic force microscopy, surface force apparatus, and so on, have been used for studying the ion structuring of the IL interface layer. Molecular Dynamics simulations have been widely used to investigate the microscopic behavior of the IL interface layer. To interpret and clarify the IL structure and dynamics as well as to predict their properties, it is always beneficial to combine both experiments and simulations as close as possible. In another theoretical model development to bridge the structure and properties of the IL interface layer with performance, thermodynamic prediction & property modeling has been demonstrated as an effective tool to add the properties and function of the studied nanomaterials. Herein, we present recent findings from applying the multiscale triangle experiment-simulation-thermodynamic modeling in the studies of ion structuring of ILs in the vicinity of solid surfaces, as well as how it qualitatively and quantitatively correlates to the overall ILs properties, performance, and function. We introduce the most common techniques behind experiment-simulation-thermodynamic modeling and how they are applied for studying the IL interface layer structuring, and we highlight the possibilities of the IL interface layer structuring in applications such as lubrication and energy storage. Integrative experiment-simulation-thermodynamic modeling is highly demanded for qualitatively and quantitatively correlating the ionic liquids interface layer structuring to the overall properties, performance, and function.

  • 6. Andersson, Annika
    et al.
    Remnestål, Julia
    Nellgård, Bengt
    Vunk, Helian
    Kotol, David
    Edfors, Fredrik
    Uhlén, Mathias
    Schwenk, Jochen M.
    Ilag, Leopold L.
    Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
    Zetterberg, Henrik
    Blennow, Kaj
    Månberg, Anna
    Nilsson, Peter
    Fredolini, Claudia
    Development of parallel reaction monitoring assays for cerebrospinal fluid proteins associated with Alzheimer's disease2019In: Clinica Chimica Acta, ISSN 0009-8981, E-ISSN 1873-3492, Vol. 494, p. 79-93Article in journal (Refereed)
    Abstract [en]

    Detailed knowledge of protein changes in cerebrospinal fluid (CSF) across healthy and diseased individuals would provide a better understanding of the onset and progression of neurodegenerative disorders. In this study, we selected 20 brain-enriched proteins previously identified in CSF by antibody suspension bead arrays (SBA) to be potentially biomarkers for Alzheimer's disease (AD) and verified these using an orthogonal approach. We examined the same set of 94 CSF samples from patients affected by AD (including preclinical and prodromal), mild cognitive impairment (MCI), non-AD dementia and healthy individuals, which had previously been analyzed by SBA. Twenty-eight parallel reaction monitoring (PRM) assays were developed and 13 of them could be validated for protein quantification. Antibody profiles were verified by PRM. For seven proteins, the antibody profiles were highly correlated with the PRM results (r > 0.7) and GAP43, VCAM1 and PSAP were identified as potential markers of preclinical AD. In conclusion, we demonstrate the usefulness of targeted mass spectrometry as a tool for the orthogonal verification of antibody profiling data, suggesting that these complementary methods can be successfully applied for comprehensive exploration of CSF protein levels in neurodegenerative disorders.

  • 7.
    Andreo, Pedro
    et al.
    Stockholm University, Faculty of Science, Department of Physics.
    Wulff, Joerg
    Burns, David T.
    Palmans, Hugo
    Consistency in reference radiotherapy dosimetry: resolution of an apparent conundrum when Co-60 is the reference quality for charged-particle and photon beams2013In: Physics in Medicine and Biology, ISSN 0031-9155, E-ISSN 1361-6560, Vol. 58, no 19, p. 6593-6621Article in journal (Refereed)
    Abstract [en]

    Substantial changes in ion chamber perturbation correction factors in Co-60 gamma-rays, suggested by recent Monte Carlo (MC) calculations, would cause a decrease of about 1.5% in the reference dosimetry of all types of charged particles (electrons, protons and heavier ions) based on calculated k(Q) values. It has gone largely unnoticed that the ratio of calibration coefficients N-D,N-w,N-Co60 and N-K,N-air,N-Co60 yields an experimental value of F-ch,F-Co60 = (s(w-air) pch)(Co60) through N-D,N-air,N-Co60. Coefficients provided by the IAEA and traceable to the BIPM for 91 NE-2571 chambers result in an average F-ch,F-Co60 which is compared with published (and new) MC simulations and with the value in IAEA TRS-398. It is shown that TRS-398 agrees within 0.12% with the experimental F-ch,F-Co60. The 1.5% difference resulting from MC calculations (1.1% for the new simulations) cannot be justified using current fundamental data and BIPM standards if consistency in the entire dosimetry chain is sought. For photons, MC k(Q) factors are compared with TRS-398. Using the same uncertainty for W-air, the two sets of data overlap considerably. Experimental k(Q) values from standards laboratories lie between the two sets of calculated values, showing no preference for one set over the other. Observed chamber-to-chamber differences, that include the effect of waterproof sleeves (also seen for Co-60), justify the recommendation in TRS-398 for k(Q) values specifically measured for the user chamber. Current developments on I-values for the stopping powers of water and graphite are presented. A weighted average I-water = 78 +/- 2 eV is obtained from published experimental and DRF-based values; this would decrease sw-air for all types of radiotherapy beams between 0.3% and 0.6%, and would consequently decrease the MC derived F-ch,F-Co60. The implications of a recent proposal for I-graphite = 81 eV are analysed, resulting in a potential decrease of 0.7% in N-K,N-air,N-Co60 which would raise the experimental F-ch,F-Co60; this would result in an increase of about 0.8% in the current TRS-398 value when referred to the BIPM standards. MC derived F-ch,F-Co60 using new stopping powers would then agree at a level of 0.1% with the experimental value, confirming the need for consistency in the dosimetry chain data. Should world average standards be used as reference, the figures would become +0.4% for TRS-398 and -0.3% for the MC calculation. F-ch,F-Q calculated for megavoltage photons using new stopping powers would decrease by between 0.2% and 0.5%. When they enter as a ratios in k(Q), differences with MC values based on current key data would be within 0.2% but their discrepancy with k(Q) experimental photon values remains unresolved. For protons the new data would require an increase in W-air,W-Q of about 0.6%, as this is inferred from a combination of calorimetry and ionometry. This consistent scenario would leave unchanged the current TRS-398 k(Q) (NE-2571) data for protons, as well as for ions heavier than protons unless new independent W-air,W-Q values become available. Also in these advanced radiotherapy modalities, the need for maintaining data consistency in an analysis that unavoidably must include the complete dosimetry chain is demonstrated.

  • 8.
    Baars, Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Protein targeting, translocation and insertion in Escherichia coli: Proteomic analysis of substrate-pathway relationships2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Approximately 10% of the open reading frames in the genome of the Gram-negative bacterium E. coli encodes secretory proteins, and 20% encodes integral inner membrane proteins (IMPs). These proteins are sorted to their correct cellular compartments (the periplasm and the outer and inner membranes) by specialized targeting and translocation/insertion systems. So far, a very limited set of model proteins have been used to study proteins sorting requirements in E. coli. The main objective of all the papers presented in this thesis was to determine the targeting and translocation/insertion requirements of more E. coli proteins. In papers I and II, this was done using focused approaches. Selected model proteins (lipoproteins and putative outer membrane proteins) were expressed from plasmids and their targeting and translocation were analysed in vitro by crosslinking experiments and/or in vivo by pulse-chase analysis in different E. coli mutant strains. In papers III a comparative sub-proteome analysis was carried out to define the role of the cytoplasmic chaperone SecB in protein targeting. In paper IV, a similar approach was used to study how protein translocation and insertion is affected upon depletion of the essential Sec-translocon component SecE. The ‘global’ approach used in paper III and IV allowed us to study protein targeting and translocation/insertion requirements on a proteome level. This led to the identification of several novel SecB substrates and a large number of potential Sec-translocon independent IMPs.

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  • 9.
    Bajinskis, Ainars
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Natarajan, Adayapalam T.
    Erixon, Klaus
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Harms-Ringdahl, Mats
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    DNA double strand breaks induced by the indirect effect of radiation are more efficiently repaired by non-homologous end joining compared to homologous recombination repair2013In: Mutation research. Genetic toxicology and environmental mutagenesis, ISSN 1383-5718, E-ISSN 1879-3592, Vol. 756, no 1-2, p. 21-29Article in journal (Refereed)
    Abstract [en]

    The aim of this study was to investigate the relative involvement of three major DNA repair pathways, i.e., non-homologous end joining (NHEJ), homologous recombination (HRR) and base excision (BER) in repair of DNA lesions of different complexity induced by low- or high-LET radiation with emphasis on the contribution of the indirect effect of radiation for these radiation qualities. A panel of DNA repair-deficient CHO cell lines was irradiated by Cs-137 gamma-rays or radon progeny alpha-particles. Irradiation was also performed in the presence of 2 M DMSO to reduce the indirect effect of radiation and the complexity of the DNA damage formed. Clonogenic survival and micronucleus assays were used to estimate efficiencies of the different repair pathways for DNA damages produced by direct and indirect effects. Removal of the indirect effect of low-LET radiation by DMSO increased clonogenic survival and decreased MN formation for all cell lines investigated. A direct contribution of the indirect effect of radiation to DNA base damage was suggested by the significant protection by DMSO seen for the BER deficient cell line. Lesions formed by the indirect effect are more readily repaired by the NHEJ pathway than by HRR after irradiation with gamma-rays or alpha-particles as evaluated by cell survival and the yields of MN. The results obtained with BER- and NHEJ-deficient cells suggest that the indirect effect of radiation contributes significantly to the formation of repair substrates for these pathways.

  • 10.
    Bakali, Amin
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Herman, Maria Dolores
    Johnson, Kenneth A.
    Kelly, Amélie A.
    Wieslander, Åke
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hallberg, B. M.
    Nordlund, Pär
    Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site2007In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 282, no 27, p. 19644-19652Article in journal (Refereed)
    Abstract [en]

    The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.

  • 11. Ball, Frank
    et al.
    Britton, Tom
    Stockholm University, Faculty of Science, Department of Mathematics.
    Sirl, David
    Household epidemic models with varying infection response2011In: Journal of Mathematical Biology, ISSN 0303-6812, E-ISSN 1432-1416, Vol. 63, no 2, p. 309-337Article in journal (Refereed)
    Abstract [en]

    This paper is concerned with SIR (susceptible -> infected -> removed) household epidemic models in which the infection response may be either mild or severe, with the type of response also affecting the infectiousness of an individual. Two different models are analysed. In the first model, the infection status of an individual is predetermined, perhaps due to partial immunity, and in the second, the infection status of an individual depends on the infection status of its infector and on whether the individual was infected by a within- or between-household contact. The first scenario may be modelled using a multitype household epidemic model, and the second scenario by a model we denote by the infector-dependent-severity household epidemic model. Large population results of the two models are derived, with the focus being on the distribution of the total numbers of mild and severe cases in a typical household, of any given size, in the event that the epidemic becomes established. The aim of the paper is to investigate whether it is possible to determine which of the two underlying explanations is causing the varying response when given final size household outbreak data containing mild and severe cases. We conduct numerical studies which show that, given data on sufficiently many households, it is generally possible to discriminate between the two models by comparing the Kullback-Leibler divergence for the two fitted models to these data.

  • 12.
    Baumgarten, Thomas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Löw, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bonde, Ida
    Herrgård, Markus J.
    Heipieper, Hermann J.
    Nørholm, Morten H. H.
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 45089Article in journal (Refereed)
    Abstract [en]

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

  • 13. Bebbington, Jan
    et al.
    Blasiak, Robert
    Stockholm University, Faculty of Science, Stockholm Resilience Centre.
    Larrinaga, Carlos
    Russell, Shona
    Sobkowiak, Madlen
    Jouffray, Jean-Baptiste
    Stockholm University, Faculty of Science, Stockholm Resilience Centre.
    Österblom, Henrik
    Stockholm University, Faculty of Science, Stockholm Resilience Centre.
    Shaping nature outcomes in corporate settings2024In: Philosophical Transactions of the Royal Society of London. Biological Sciences, ISSN 0962-8436, E-ISSN 1471-2970, Vol. 379, no 1903, article id 20220325Article in journal (Refereed)
    Abstract [en]

    Transnational companies have substantive impacts on nature: a hallmark of living in the Anthropocene. Understanding these impacts through company provision of information is a precursor to holding them accountable for nature outcomes. The effect of increasing disclosures (of varying quality) is predicated on 'information governance', an approach that uses disclosure requirements to drive company behaviour. However, its efficacy is not guaranteed. We argue that three conditions are required before disclosures have the possibility to shape nature outcomes, namely: (1) radical traceability that links company actions to outcomes in particular settings; (2) developing organizational routines, tools and approaches that translate strategic intent to on-the-ground behaviour; and (3) mobilizing and aligning financial actors with corporate nature ambitions. While disclosure is key to each of these conditions, its limits must be taken into account and it must be nested in governance approaches that shape action, not just reporting.This article is part of the theme issue 'Bringing nature into decision-making'.

  • 14. Bergh, Johan
    et al.
    Zetterstrom, Per
    Andersen, Peter M.
    Brannstrom, Thomas
    Graffmo, Karin S.
    Jonsson, P. Andreas
    Lang, Lisa
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Danielsson, Jens
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oliveberg, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Marklund, Stefan L.
    Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 14, p. 4489-4494Article in journal (Refereed)
    Abstract [en]

    Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

  • 15.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dual Targeting of Proteins to Mitochondria and Chloroplasts2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The vast majority of mitochondrial and chloroplastic proteins are nuclear encoded, synthesized in the cytosol and imported into the respective organelle using an N-terminal extension, the targeting peptide (TP). After import into the organelle, the TP is cleaved off and degraded by the Presequence protease (PreP). The import process is thought to be highly specific, however there is a group of proteins that are localised to both mitochondria and chloroplasts, using an ambiguous, dual targeting peptide (dTP). The aim of this thesis was to investigate targeting properties of dTPs. Analysis of the amino acid content of all currently known dually targeted proteins revealed that the dTPs are enriched in hydroxylated, hydrophobic and positively charged residues, lacking acidic residues, whereas the content of serine, arginine and proline is intermediary in comparison to the mitochondrial and chloroplastic TPs. dTPs do not form amphiphilic a-helices, characteristic of the mitochondrial TPs, but the helical structure can be induced in membrane mimetic environment, as revealed by spectroscopic studies of a dTP of an aminoacyl- tRNA-synthetase (aaRS). In vitro and in vivo import experiments of fusion constructs containing N-terminal truncations of seven aaRS-dTPs coupled to green fluorescent protein (GFP) demonstrated different organisation of targeting determinants showing that the N-terminal portion of dTPs was crucial for import into both organelles or at least one organelle for different constructs. In addition, studies of targeting capacity of the TPs of PreP homologues from plant, mammal and yeast (AtPreP, hPreP and Mop112) showed species dependent intra-mitochondrial localisation of the coupled GFP and demonstrated functional complementation of an intermembrane space located Mop112 with a matrix located AtPreP. The studies presented here contribute to understanding of the intracellular and intra-mitochondrial sorting process of proteins in the eukaryotic cell.

  • 16. Bestas, Burcu
    et al.
    Moreno, Pedro M. D.
    Blomberg, K. Emelie M.
    Mohammad, Dara K.
    Saleh, Amer F.
    Sutlu, Tolga
    Nordin, Joel Z.
    Guterstam, Peter
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Gustafsson, Manuela O.
    Kharazi, Shabnam
    Piatosa, Barbara
    Roberts, Thomas C.
    Behlke, Mark A.
    Wood, Matthew J. A.
    Gait, Michael J.
    Lundin, Karin E.
    EL Andaloussi, Samir
    Mansson, Robert
    Berglof, Anna
    Wengel, Jesper
    Smith, C. I. Edvard
    Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model2014In: Journal of Clinical Investigation, ISSN 0021-9738, E-ISSN 1558-8238, Vol. 124, no 9, p. 4067-4081Article in journal (Refereed)
    Abstract [en]

    X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.

  • 17.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuhn, Claus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Berglund, Anna-Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Roth, Christian
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The role of the N-terminal domain of chloroplast targeting peptides in organellar protein import and miss-sorting2006In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 580, no 16, p. 3966-3972Article in journal (Refereed)
    Abstract [en]

    We have analysed 385 mitochondrial and 567 chloroplastic signal sequences of proteins found in the organellar proteomes of Arabidopsis thaliana. Despite overall similarities, the first 16 residues of transit peptides differ remarkably. To test the hypothesis that the N-terminally truncated transit peptides would redirect chloroplastic precursor proteins to mitochondria, we studied import of the N-terminal deletion mutants of ELIP, PetC and Lhcb2.1. The results show that the deletion mutants were neither imported into chloroplasts nor miss-targeted to mitochondria in vitro and in vivo, showing that the entire transit peptide is necessary for correct targeting as well as miss-sorting.

  • 18.
    Bhushan, Shashi
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pavlov, Pavel F
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rudhe, Charlotta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    In vitro and in vivo methods to study protein import into plant mitochondria.2007In: Methods Mol Biol, ISSN 1064-3745, Vol. 390, p. 131-50Article in journal (Refereed)
    Abstract [en]

    Plant mitochondria contain about 1000 proteins, 90-99% of which in different plant species are nuclear encoded, synthesized on cytosolic polyribosomes, and imported into the organelle. Most of the nuclear-encoded proteins are synthesized as precursors containing an N-terminal extension called a presequence or targeting peptide that directs the protein to the mitochondria. Here we describe in vitro and in vivo methods to study mitochondrial protein import in plants. In vitro synthesized precursor proteins can be imported in vitro into isolated mitochondria (single organelle import). However, missorting of chloroplast precursors in vitro into isolated mitochondria has been observed. A novel dual import system for simultaneous import of proteins into isolated mitochondria and chloroplasts followed by reisolation of the organelles is superior over the single import system as it abolishes the mistargeting. Precursor proteins can also be imported into the mitochondria in vivo using an intact cellular system. In vivo approaches include import of transiently expressed fusion constructs containing a presequence or a full-length precursor protein fused to a reporter gene, most commonly the green fluorescence protein (GFP) in protoplasts or in an Agrobacterium-mediated system in intact tobacco leaves.

  • 19. Bjorklund, Geir
    et al.
    Stejskal, Vera
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Urbina, Mauricio A.
    Dadar, Maryam
    Chirumbolo, Salvatore
    Mutter, Joachim
    Metals and Parkinson's Disease: Mechanisms and Biochemical Processes2018In: Current Medicinal Chemistry, ISSN 0929-8673, E-ISSN 1875-533X, Vol. 25, no 19, p. 2198-2214Article, review/survey (Refereed)
    Abstract [en]

    Genetic background accounts for only 5 to 10% of the reported cases of Parkinson's disease (PD), while the remaining cases are of unknown etiology. It is believed that environmental factors may be involved in the causality of a large proportion of PD cases. Several PD genes are activated by xenobiotic exposure, and a link between pesticide exposure and PD has been demonstrated. Many epidemiological studies have shown an association between PD and exposure to metals such as mercury, lead, manganese, copper, iron, aluminum, bismuth, thallium, and zinc. This review explores the biological effects, the pathogenetic processes, genetic susceptibilities to metals as well as examining future strategies for PD treatment, such as chelation therapy.

  • 20.
    Björkander, Sophia
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hell, Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Johansson, Maria A.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Mata Forsberg, Manuel
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lasaviciute, Gintare
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Roos, Stefan
    Holmlund, Ulrika
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sverremark-Ekström, Eva
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Staphylococcus aureus-derived factors induce IL-10, IFN-gamma and IL-17A-expressing FOXP3(+)CD161(+) T-helper cells in a partly monocyte-dependent manner2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 22083Article in journal (Refereed)
    Abstract [en]

    Staphylococcus aureus (S. aureus) is a human pathogen as well as a frequent colonizer of skin and mucosa. This bacterium potently activates conventional T-cells through superantigens and it is suggested to induce T-cell cytokine-production as well as to promote a regulatory phenotype in T-cells in order to avoid clearance. This study aimed to investigate how S. aureus impacts the production of regulatory and pro-inflammatory cytokines and the expression of CD161 and HELIOS by peripheral CD4(+)FOXP3(+) T-cells. Stimulation of PBMC with S. aureus 161:2-cell free supernatant (CFS) induced expression of IL-10, IFN-gamma and IL-17A in FOXP3(+) cells. Further, CD161 and HELIOS separated the FOXP3(+) cells into four distinct populations regarding cytokine-expression. Monocyte-depletion decreased S. aureus 161:2-induced activation of FOXP3(+) cells while pre-stimulation of purified monocytes with S. aureus 161:2-CFS and subsequent co-culture with autologous monocyte-depleted PBMC was sufficient to mediate activation of FOXP3(+) cells. Together, these data show that S. aureus potently induces FOXP3(+) cells and promotes a diverse phenotype with expression of regulatory and pro-inflammatory cytokines connected to increased CD161-expression. This could indicate potent regulation or a contribution of FOXP3(+) cells to inflammation and repression of immune-suppression upon encounter with S. aureus.

  • 21.
    Björklund, Asa K
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Light, Sara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hedin, Linnea
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Quantitative assessment of the structural bias in protein-protein interaction assays.2008In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 8, no 22, p. 4657-46667Article in journal (Refereed)
    Abstract [en]

    With recent publications of several large-scale protein-protein interaction (PPI) studies, the realization of the full yeast interaction network is getting closer. Here, we have analysed several yeast protein interaction datasets to understand their strengths and weaknesses. In particular, we investigate the effect of experimental biases on some of the protein properties suggested to be enriched in highly connected proteins. Finally, we use support vector machines (SVM) to assess the contribution of these properties to protein interactivity. We find that protein abundance is the most important factor for detecting interactions in tandem affinity purifications (TAP), while it is of less importance for Yeast Two Hybrid (Y2H) screens. Consequently, sequence conservation and/or essentiality of hubs may be related to their high abundance. Further, proteins with disordered structure are over-represented in Y2H screens and in one, but not the other, large-scale TAP assay. Hence, disordered regions may be important both in transient interactions and interactions in complexes. Finally, a few domain families seem to be responsible for a large part of all interactions. Most importantly, we show that there are method-specific biases in PPI experiments. Thus, care should be taken before drawing strong conclusions based on a single dataset.

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  • 22.
    Björklund, Åsa K.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ekman, Diana
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Elofsson, Arne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Expansion of Protein Domain Repeats2006In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 2, no 8, p. 959-970Article in journal (Refereed)
    Abstract [en]

    Many proteins, especially in eukaryotes, contain tandem repeats of several domains from the same family. These repeats have a variety of binding properties and are involved in protein-protein interactions as well as binding to other ligands such as DNA and RNA. The rapid expansion of protein domain repeats is assumed to have evolved through internal tandem duplications. However, the exact mechanisms behind these tandem duplications are not well-understood. Here, we have studied the evolution, function, protein structure, gene structure, and phylogenetic distribution of domain repeats. For this purpose we have assigned Pfam-A domain families to 24 proteomes with more sensitive domain assignments in the repeat regions. These assignments confirmed previous findings that eukaryotes, and in particular vertebrates, contain a much higher fraction of proteins with repeats compared with prokaryotes. The internal sequence similarity in each protein revealed that the domain repeats are often expanded through duplications of several domains at a time, while the duplication of one domain is less common. Many of the repeats appear to have been duplicated in the middle of the repeat region. This is in strong contrast to the evolution of other proteins that mainly works through additions of single domains at either terminus. Further, we found that some domain families show distinct duplication patterns, e. g., nebulin domains have mainly been expanded with a unit of seven domains at a time, while duplications of other domain families involve varying numbers of domains. Finally, no common mechanism for the expansion of all repeats could be detected. We found that the duplication patterns show no dependence on the size of the domains. Further, repeat expansion in some families can possibly be explained by shuffling of exons. However, exon shuffling could not have created all repeats.

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  • 23. Brechmann, Nils A.
    et al.
    Eriksson, Per-Olov
    Eriksson, Kristofer
    Oscarsson, Sven
    Stockholm University, Faculty of Science, Department of Organic Chemistry.
    Buijs, Jos
    Shokri, Atefeh
    Hjälm, Göran
    Chotteau, Véronique
    Pilot-scale process for magnetic bead purification of antibodies directly from non-clarified CHO cell culture2019In: Biotechnology progress (Print), ISSN 8756-7938, E-ISSN 1520-6033, Vol. 35, no 3, article id e2775Article in journal (Refereed)
    Abstract [en]

    High capacity magnetic protein A agarose beads, LOABeads PrtA, were used in the development of a new process for affinity purification of monoclonal antibodies (mAbs) from non-clarified CHO cell broth using a pilot-scale magnetic separator. The LOABeads had a maximum binding capacity of 65 mg/mL and an adsorption capacity of 25-42 mg IgG/mL bead in suspension for an IgG concentration of 1 to 8 g/L. Pilot-scale separation was initially tested in a mAb capture step from 26 L clarified harvest. Small-scale experiments showed that similar mAb adsorptions were obtained in cell broth containing 40 x 10(6) cells/mL as in clarified supernatant. Two pilot-scale purification runs were then performed on non-clarified cell broth from fed-batch runs of 16 L, where a rapid mAb adsorption >= 96.6% was observed after 1 h. This process using 1 L of magnetic beads had an overall mAb yield of 86% and 16 times concentration factor. After this single protein A capture step, the mAb purity was similar to the one obtained by column chromatography, while the host cell protein content was very low, <10 ppm. Our results showed that this magnetic bead mAb purification process, using a dedicated pilot-scale separation device, was a highly efficient single step, which directly connected the culture to the downstream process without cell clarification. Purification of mAb directly from non-clarified cell broth without cell separation can provide significant savings in terms of resources, operation time, and equipment, compared to legacy procedure of cell separation followed by column chromatography step.

  • 24. Brownstein, Catherine A.
    et al.
    Beggs, Alan H.
    Homer, Nils
    Merriman, Barry
    Yu, Timothy W.
    Flannery, Katherine C.
    DeChene, Elizabeth T.
    Towne, Meghan C.
    Savage, Sarah K.
    Price, Emily N.
    Holm, Ingrid A.
    Luquette, Lovelace J.
    Lyon, Elaine
    Majzoub, Joseph
    Neupert, Peter
    McCallie, David, Jr.
    Szolovits, Peter
    Willard, Huntington F.
    Mendelsohn, Nancy J.
    Temme, Renee
    Finkel, Richard S.
    Yum, Sabrina W.
    Medne, Livija
    Sunyaev, Shamil R.
    Adzhubey, Ivan
    Cassa, Christopher A.
    de Bakker, Paul I. W.
    Duzkale, Hatice
    Dworzynski, Piotr
    Fairbrother, William
    Francioli, Laurent
    Funke, Birgit H.
    Giovanni, Monica A.
    Handsaker, Robert E.
    Lage, Kasper
    Lebo, Matthew S.
    Lek, Monkol
    Leshchiner, Ignaty
    MacArthur, Daniel G.
    McLaughlin, Heather M.
    Murray, Michael F.
    Pers, Tune H.
    Polak, Paz P.
    Raychaudhuri, Soumya
    Rehm, Heidi L.
    Soemedi, Rachel
    Stitziel, Nathan O.
    Vestecka, Sara
    Supper, Jochen
    Gugenmus, Claudia
    Klocke, Bernward
    Hahn, Alexander
    Schubach, Max
    Menzel, Mortiz
    Biskup, Saskia
    Freisinger, Peter
    Deng, Mario
    Braun, Martin
    Perner, Sven
    Smith, Richard J. H.
    Andorf, Janeen L.
    Huang, Jian
    Ryckman, Kelli
    Sheffield, Val C.
    Stone, Edwin M.
    Bair, Thomas
    Black-Ziegelbein, E. Ann
    Braun, Terry A.
    Darbro, Benjamin
    DeLuca, Adam P.
    Kolbe, Diana L.
    Scheetz, Todd E.
    Shearer, Aiden E.
    Sompallae, Rama
    Wang, Kai
    Bassuk, Alexander G.
    Edens, Erik
    Mathews, Katherine
    Moore, Steven A.
    Shchelochkov, Oleg A.
    Trapane, Pamela
    Bossler, Aaron
    Campbell, Colleen A.
    Heusel, Jonathan W.
    Kwitek, Anne
    Maga, Tara
    Panzer, Karin
    Wassink, Thomas
    Van Daele, Douglas
    Azaiez, Hela
    Booth, Kevin
    Meyer, Nic
    Segal, Michael M.
    Williams, Marc S.
    Tromp, Gerard
    White, Peter
    Corsmeier, Donald
    Fitzgerald-Butt, Sara
    Herman, Gail
    Lamb-Thrush, Devon
    McBride, Kim L.
    Newsom, David
    Pierson, Christopher R.
    Rakowsky, Alexander T.
    Maver, Ales
    Lovrecic, Luca
    Palandacic, Anja
    Peterlin, Borut
    Torkamani, Ali
    Wedell, Anna
    Huss, Mikael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Alexeyenko, Andrey
    Lindvall, Jessica M.
    Magnusson, Mans
    Nilsson, Daniel
    Stranneheim, Henrik
    Taylan, Fulya
    Gilissen, Christian
    Hoischen, Alexander
    van Bon, Bregje
    Yntema, Helger
    Nelen, Marcel
    Zhang, Weidong
    Sager, Jason
    Zhang, Lu
    Blair, Kathryn
    Kural, Deniz
    Cariaso, Michael
    Lennon, Greg G.
    Javed, Asif
    Agrawal, Saloni
    Ng, Pauline C.
    Sandhu, Komal S.
    Krishna, Shuba
    Veeramachaneni, Vamsi
    Isakov, Ofer
    Halperin, Eran
    Friedman, Eitan
    Shomron, Noam
    Glusman, Gustavo
    Roach, Jared C.
    Caballero, Juan
    Cox, Hannah C.
    Mauldin, Denise
    Ament, Seth A.
    Rowen, Lee
    Richards, Daniel R.
    San Lucas, F. Anthony
    Gonzalez-Garay, Manuel L.
    Caskey, C. Thomas
    Bai, Yu
    Huang, Ying
    Fang, Fang
    Zhang, Yan
    Wang, Zhengyuan
    Barrera, Jorge
    Garcia-Lobo, Juan M.
    Gonzalez-Lamuno, Domingo
    Llorca, Javier
    Rodriguez, Maria C.
    Varela, Ignacio
    Reese, Martin G.
    De la Vega, Francisco M.
    Kiruluta, Edward
    Cargill, Michele
    Hart, Reece K.
    Sorenson, Jon M.
    Lyon, Gholson J.
    Stevenson, David A.
    Bray, Bruce E.
    Moore, Barry M.
    Eilbeck, Karen
    Yandell, Mark
    Zhao, Hongyu
    Hou, Lin
    Chen, Xiaowei
    Yan, Xiting
    Chen, Mengjie
    Li, Cong
    Yang, Can
    Gunel, Murat
    Li, Peining
    Kong, Yong
    Alexander, Austin C.
    Albertyn, Zayed I.
    Boycott, Kym M.
    Bulman, Dennis E.
    Gordon, Paul M. K.
    Innes, A. Micheil
    Knoppers, Bartha M.
    Majewski, Jacek
    Marshall, Christian R.
    Parboosingh, Jillian S.
    Sawyer, Sarah L.
    Samuels, Mark E.
    Schwartzentruber, Jeremy
    Kohane, Isaac S.
    Margulies, David M.
    An international effort towards developing standards for best practices in analysis, interpretation and reporting of clinical genome sequencing results in the CLARITY Challenge2014In: Genome Biology, ISSN 1465-6906, E-ISSN 1474-760X, Vol. 15, no 3, article id R53Article in journal (Refereed)
    Abstract [en]

    Background: There is tremendous potential for genome sequencing to improve clinical diagnosis and care once it becomes routinely accessible, but this will require formalizing research methods into clinical best practices in the areas of sequence data generation, analysis, interpretation and reporting. The CLARITY Challenge was designed to spur convergence in methods for diagnosing genetic disease starting from clinical case history and genome sequencing data. DNA samples were obtained from three families with heritable genetic disorders and genomic sequence data were donated by sequencing platform vendors. The challenge was to analyze and interpret these data with the goals of identifying disease-causing variants and reporting the findings in a clinically useful format. Participating contestant groups were solicited broadly, and an independent panel of judges evaluated their performance. Results: A total of 30 international groups were engaged. The entries reveal a general convergence of practices on most elements of the analysis and interpretation process. However, even given this commonality of approach, only two groups identified the consensus candidate variants in all disease cases, demonstrating a need for consistent fine-tuning of the generally accepted methods. There was greater diversity of the final clinical report content and in the patient consenting process, demonstrating that these areas require additional exploration and standardization. Conclusions: The CLARITY Challenge provides a comprehensive assessment of current practices for using genome sequencing to diagnose and report genetic diseases. There is remarkable convergence in bioinformatic techniques, but medical interpretation and reporting are areas that require further development by many groups.

  • 25.
    Brändén, Magnus
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Electron and proton transfer in cytochrome c oxidase2003Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This doctoral thesis describes results from studies on the mechanisms by which the enzyme cytochrome c oxidase catalyses the reduction of oxygen to water. Cytochrome c oxidase is the last integral enzyme complex of the socalled respiratory chain in the mitochondrial inner membrane (plasma membrane of bacteria). The reaction catalysed by cytochrome c oxidase produces a charge separation across the membrane by two separate mechanisms. The protons and the electrons needed to reduce oxygen to water are supplied from different sides of the membrane, and the energy released in this reaction is used by cytochrome c oxidase to pump protons against the electro-chemical gradient across the membrane. Another integral enzyme, ATP-synthase, utilises this gradient for synthesis of ATP, which in turn is used in many of the energyrequiring processes of a cell. Cytochrome c oxidase holds four redox-active metal centres that mediate electron transfer to, and reduction of the O2-molecule. One part of the thesis concerns the rates of electron transfer as well as the redox equilibrium between the metal sites (papers II and IV).

    Since the oxygen chemistry takes place in the membrane-spanning part of the enzyme it is equipped with two proton-transfer pathways that lead from the bulk solution to the active site. A second part of the thesis concerns the location of the entry-point of one of these pathways as well as the role of this pathway during the catalytic cycle (papers I and III).

    The third part of the thesis concerns the mechanism by which the enzymecouples the O2-chemistry to proton pumping (paper V).

  • 26.
    Buzzao, Davide
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Castresana-Aguirre, Miguel
    Guala, Dimitri
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Sonnhammer, Erik L. L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    TOPAS, a network-based approach to detect disease modules in a top-down fashion 2022In: NAR Genomics and Bioinformatics, E-ISSN 2631-9268, Vol. 4, no 4, article id lqac093Article in journal (Refereed)
    Abstract [en]

    A vast scenario of potential disease mechanisms and remedies is yet to be discovered. The field of Network Medicine has grown thanks to the massive amount of high-throughput data and the emerging evidence that disease-related proteins form ‘disease modules’. Relying on prior disease knowledge, network-based disease module detection algorithms aim at connecting the list of known disease associated genes by exploiting interaction networks. Most existing methods extend disease modules by iteratively adding connector genes in a bottom-up fashion, while top-down approaches remain largely unexplored. We have created TOPAS, an iterative approach that aims at connecting the largest number of seed nodes in a top-down fashion through connectors that guarantee the highest flow of a Random Walk with Restart in a network of functional associations. We used a corpus of 382 manually selected functional gene sets to benchmark our algorithm against SCA, DIAMOnD, MaxLink and ROBUST across four interactomes. We demonstrate that TOPAS outperforms competing methods in terms of Seed Recovery Rate, Seed to Connector Ratio and consistency during module detection. We also show that TOPAS achieves competitive performance in terms of biological relevance of detected modules and scalability. 

  • 27.
    Bäckman, Hans G
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pessoa, João
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eneqvist, Therese
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Glaser, Elzbieta
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Binding of divalent cations is essential for the activity of the organellar peptidasome in Arabidopsis thaliana, AtPreP.2009In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 583, no 17, p. 2727-33Article in journal (Refereed)
    Abstract [en]

    The dual-targeted mitochondrial and chloroplastic zinc metallooligopeptidase from Arabidopsis, AtPreP, functions as a peptidasome that degrades targeting peptides and other small unstructured peptides. In addition to Zn located in the catalytic site, AtPreP also contains two Mg-binding sites. We have investigated the role of Mg-binding using AtPreP variants, in which one or both sites were rendered unable to bind Mg(2+). Our results show that metal binding besides that of the active site is crucial for AtPreP proteolysis, particularly the inner site appears essential for normal proteolytic function. This is also supported by its evolutionary conservation among all plant species of PreP.

  • 28. Cappellini, Francesca
    et al.
    Di Bucchianico, Sebastiano
    Karri, Venkatanaidu
    Latvala, Siiri
    Stockholm University, Faculty of Science, Department of Environmental Science.
    Malmlof, Maria
    Kippler, Maria
    Elihn, Karine
    Stockholm University, Faculty of Science, Department of Environmental Science.
    Hedberg, Jonas
    Wallinder, Inger Odnevall
    Gerde, Per
    Karlsson, Hanna L.
    Dry Generation of CeO2 Nanoparticles and Deposition onto a Co-Culture of A549 and THP-1 Cells in Air-Liquid Interface-Dosimetry Considerations and Comparison to Submerged Exposure2020In: Nanomaterials, E-ISSN 2079-4991, Vol. 10, no 4, article id 618Article in journal (Refereed)
    Abstract [en]

    Relevant in vitro assays that can simulate exposure to nanoparticles (NPs) via inhalation are urgently needed. Presently, the most common method employed is to expose lung cells under submerged conditions, but the cellular responses to NPs under such conditions might differ from those observed at the more physiological air-liquid interface (ALI). The aim of this study was to investigate the cytotoxic and inflammatory potential of CeO2 NPs (NM-212) in a co-culture of A549 lung epithelial cells and differentiated THP-1 cells in both ALI and submerged conditions. Cellular dose was examined quantitatively using inductively coupled plasma mass spectrometry (ICP-MS). The role of serum and LPS-priming for IL-1 beta release was further tested in THP-1 cells in submerged exposure. An aerosol of CeO2 NPs was generated by using the PreciseInhale (R) system, and NPs were deposited on the co-culture using XposeALI (R). No or minor cytotoxicity and no increased release of inflammatory cytokines (IL-1 beta, IL-6, TNF alpha, MCP-1) were observed after exposure of the co-culture in ALI (max 5 mu g/cm(2)) or submerged (max 22 mu g/cm(2)) conditions. In contrast, CeO2 NPs cause clear IL-1 beta release in monocultures of macrophage-like THP-1, independent of the presence of serum and LPS-priming. This study demonstrates a useful approach for comparing effects at various in-vitro conditions.

  • 29. Carter, Victoria
    et al.
    Underhill, Ann
    Baber, Ibrahima
    Sylla, Lakamy
    Baby, Mounirou
    Larget-Thiery, Isabelle
    Zettor, Agnès
    Bourgouin, Catherine
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Otvos, Laszlo
    Wade, John D.
    Coulibaly, Mamadou B.
    Traore, Sekou F.
    Tripet, Frederic
    Eggleston, Paul
    Hurd, Hilary
    Killer bee molecules: antimicrobial peptides as effector molecules to target sporogonic stages of Plasmodium2013In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 9, no 11, article id e1003790Article in journal (Refereed)
    Abstract [en]

    A new generation of strategies is evolving that aim to block malaria transmission by employing genetically modified vectors or mosquito pathogens or symbionts that express anti-parasite molecules. Whilst transgenic technologies have advanced rapidly, there is still a paucity of effector molecules with potent anti-malaria activity whose expression does not cause detrimental effects on mosquito fitness. Our objective was to examine a wide range of antimicrobial peptides (AMPs) for their toxic effects on Plasmodium and anopheline mosquitoes. Specifically targeting early sporogonic stages, we initially screened AMPs for toxicity against a mosquito cell line and P. berghei ookinetes. Promising candidate AMPs were fed to mosquitoes to monitor adverse fitness effects, and their efficacy in blocking rodent malaria infection in Anopheles stephensi was assessed. This was followed by tests to determine their activity against P. falciparum in An. gambiae, initially using laboratory cultures to infect mosquitoes, then culminating in preliminary assays in the field using gametocytes and mosquitoes collected from the same area in Mali, West Africa. From a range of 33 molecules, six AMPs able to block Plasmodium development were identified: Anoplin, Duramycin, Mastoparan X, Melittin, TP10 and Vida3. With the exception of Anoplin and Mastoparan X, these AMPs were also toxic to an An. gambiae cell line at a concentration of 25 µM. However, when tested in mosquito blood feeds, they did not reduce mosquito longevity or egg production at concentrations of 50 µM. Peptides effective against cultured ookinetes were less effective when tested in vivo and differences in efficacy against P. berghei and P. falciparum were seen. From the range of molecules tested, the majority of effective AMPs were derived from bee/wasp venoms.

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  • 30. Cerrato, Carmine Pasquale
    et al.
    Kivijärvi, Tove
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. University of Tartu, Estonia.
    Mitochondrial Targeting Probes, Drug Conjugates, and Gene Therapeutics2022In: Cell Penetrating Peptides: Methods and Protocols / [ed] Ülo Langel, New York: Humana Press, 2022, p. 429-446Chapter in book (Refereed)
    Abstract [en]

    Mitochondria represent an important drug target for many phatology, including neurodegeneration, metabolic disease, heart failure, ischemia-reperfusion injury, and cancer. Mitochondrial dysfunctions are caused by mutation in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins. Cell-penetrating peptides (CPPs) have been employed to overcome biological barriers, target this organelle, and therapeuticaly restore mitochondrial functions. Here, we describe recent methods used to deliver oligonucleotides targeting mitochondrial protein by using mitochondrial penetrating peptides. In particular, we highlight recent advances of formulated peptides/oligonucleotides nanocomplexes as a proof-of-principle for pharmaceutical form of peptide-based therapeutics. 

  • 31. 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.

  • 32.
    Chiruvella, Kishore K.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Rajaei, Naghmeh
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Jonna, Venkateswara Rao
    Hofer, Anders
    Åström, Stefan U.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Biochemical Characterization of Kat1: a Domesticated hAT-Transposase that Induces DNA Hairpin Formation and MAT-Switching2016In: Scientific Reports, E-ISSN 2045-2322, Vol. 6, article id 21671Article in journal (Refereed)
    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.

  • 33. Clausson, Carl-Magnus
    et al.
    Arngården, Linda
    Ishaq, Omer
    Klaesson, Axel
    Kühnemund, Malte
    Grannas, Karin
    Koos, Björn
    Qian, Xiaoyan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Ranefall, Petter
    Krzywkowski, Tomasz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Brismar, Hjalmar
    Nilsson, Mats
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Wählby, Carolina
    Söderberg, Ola
    Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio2015In: Scientific Reports, E-ISSN 2045-2322, Vol. 5, article id 12317Article in journal (Refereed)
    Abstract [en]

    Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes.

  • 34.
    Cumming, Alister James
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Next generation tools for microbial cell factories2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The bacterium E. coli is a popular choice for expressing recombinant proteins. It is easy to culture and many molecular tools for are available for directing protein production. When producing recombinant proteins, high yields and good quality soluble products are desirable. Poor yields or misfolded, aggregated and insoluble products are unwanted outcomes. In this thesis, existing tools have been evolved to enable increased yields of recombinant products from microbial cell factories. New tools have also been developed that help the user avoid bacterial stress and improve the quality of the recombinant proteins. 

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  • 35. Czub, Joanna
    et al.
    Banaś, Dariusz
    Braziewicz, Janusz
    Buraczewska, Iwona
    Jaskóła, Marian
    Kaźmierczak, Urszula
    Korman, Andrzej
    Lankoff, Anna
    Lisowska, Halina
    Szefliński, Zygmunt
    Wojewódzka, Maria
    Wójcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Biological effects of mixed-ion beams. Part 1: Effect of irradiation of the CHO-K1 cells with a mixed-ion beam containing the carbon and oxygen ions2018In: Applied Radiation and Isotopes, ISSN 0969-8043, E-ISSN 1872-9800, Vol. 139, p. 304-309Article in journal (Refereed)
    Abstract [en]

    Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found.

  • 36. Czub, Joanna
    et al.
    Banaś, Dariusz
    Braziewicz, Janusz
    Buraczewska, Iwona
    Jaskóła, Marian
    Kaźmierczak, Urszula
    Korman, Andrzej
    Lankoff, Anna
    Lisowska, Halina
    Szefliński, Zygmunt
    Wojewódzka, Maria
    Wójcik, Andrzej
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Jan Kochanowski University, Poland.
    Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams2019In: Applied Radiation and Isotopes, ISSN 0969-8043, E-ISSN 1872-9800, Vol. 150, p. 192-198Article in journal (Refereed)
    Abstract [en]

    The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236-300 and 461-470 keV/mu m for C-12 and O-16 ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (C-12) and 470 keV/mu m (O-16).

  • 37. Dahmane, Selma
    et al.
    Kerviel, Adeline
    Morado, Dustin R.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Shankar, Kasturika
    Ahlman, Björn
    Lazarou, Michael
    Altan-Bonnet, Nihal
    Carlson, Lars-Anders
    Membrane-assisted assembly and selective secretory autophagy of enteroviruses2022In: Nature Communications, E-ISSN 2041-1723, Vol. 13, no 1, article id 5986Article in journal (Refereed)
    Abstract [en]

    Enteroviruses are non-enveloped positive-sense RNA viruses that cause diverse diseases in humans. Their rapid multiplication depends on remodeling of cytoplasmic membranes for viral genome replication. It is unknown how virions assemble around these newly synthesized genomes and how they are then loaded into autophagic membranes for release through secretory autophagy. Here, we use cryo-electron tomography of infected cells to show that poliovirus assembles directly on replication membranes. Pharmacological untethering of capsids from membranes abrogates RNA encapsidation. Our data directly visualize a membrane-bound half-capsid as a prominent virion assembly intermediate. Assembly progression past this intermediate depends on the class III phosphatidylinositol 3-kinase VPS34, a key host-cell autophagy factor. On the other hand, the canonical autophagy initiator ULK1 is shown to restrict virion production since its inhibition leads to increased accumulation of virions in vast intracellular arrays, followed by an increased vesicular release at later time points. Finally, we identify multiple layers of selectivity in virus-induced autophagy, with a strong selection for RNA-loaded virions over empty capsids and the segregation of virions from other types of autophagosome contents. These findings provide an integrated structural framework for multiple stages of the poliovirus life cycle. Enteroviruses are non-enveloped positive-sense RNA viruses that modulate cytoplasmic membranes for replication. To enlighten how enteroviruses assemble around nascent RNA genomes and get package into autophagosomes for release, Dahmane et al. perform cryo-electron tomography of poliovirus-infected cells. They find assembly intermediates that are only present on the cytosolic side of the replication compartment and provide evidence that host factor VPS34 is involved in progression of assembly intermediates.

  • 38. Darsalia, Vladimer
    et al.
    Mansouri, Shiva
    Ortsater, Henrik
    Olverling, Anna
    Nozadze, Nino
    Kappe, Camilla
    Iverfeldt, Kerstin
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Tracy, Linda M.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Grankvist, Nina
    Sjöholm, Åke
    Patrone, Cesare
    Glucagon-like peptide-1 receptor activation reduces ischaemic brain damage following stroke in Type 2 diabetic rats2012In: Clinical Science, ISSN 0143-5221, E-ISSN 1470-8736, Vol. 122, no 9-10, p. 473-483Article in journal (Refereed)
    Abstract [en]

    Diabetes is a strong risk factor for premature and severe stroke. The GLP-IR (glucagon-like peptide-1 receptor) agonist Ex-4 (exendin-4) is a drug for the treatment of T2D (Type 2 diabetes) that may also have neuroprotective effects. The aim of the present study was to determine the efficacy of Ex-4 against stroke in diabetes by using a diabetic animal model, a drug administration paradigm and a dose that mimics a diabetic patient on Ex-4 therapy. Furthermore, we investigated inflammation and neurogenesis as potential cellular mechanisms underlying the Ex-4 efficacy. A total of seven 9-month-old Type 2 diabetic Goto-Kakizaki rats were treated peripherally for 4 weeks with Ex-4 at 0.1, 1 or 5 mu g/kg of body weight before inducing stroke by transient middle cerebral artery occlusion and for 2-4 weeks thereafter. The severity of ischaemic damage was measured by evaluation of stroke volume and by stereological counting of neurons in the striatum and cortex. We also quantitatively evaluated stroke-induced inflammation, stem cell proliferation and neurogenesis. We show a profound anti-stroke efficacy of the clinical dose of Ex-4 in diabetic rats, an arrested microglia infiltration and an increase of stroke-induced neural stem cell proliferation and neuroblast formation, while stroke-induced neurogenesis was not affected by Ex-4. The results show a pronounced anti-stroke, neuroprotective and anti-inflammatory effect of peripheral and chronic Ex-4 treatment in middle-aged diabetic animals in a preclinical setting that has the potential to mimic the clinical treatment. Our results should provide strong impetus to further investigate GLP-IR agonists for their neuroprotective action in diabetes, and for their possible use as anti-stroke medication in non-diabetic conditions.

  • 39.
    Dian, Cyril
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Adaptive Responses by Transcriptional Regulators to small molecules in Prokaryotes: Structural studies of two bacterial one-component signal transduction systems DntR and HpNikR2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Prokaryotes are continually exposed to variations in their environment. Survival in unstable milieu requires a wide range of transcriptional regulators (TRs) that respond to specific environmental and cellular signals by modulating gene expression and provide an appropriate physiological response to external stimuli. These adaptive responses to environmental signals are mostly mediated by TRs from one of two families: the single or the two component signal transduction systems (1CSTS; 2CSTS). In this thesis the structural analysis of two 1CSTS – DntR and NikR − are presented. One study was carried out to try to develop a bacterial biosensor for synthetic dinitrotulenes compounds, the other to characterise the Ni-sensing mechanism that contributes to the acid adaptation of the human pathogen Helicobacter pylori. DntR belongs to the LysR family and the crystal structures obtained have allowed the proposal a model of the interaction of DntR with salicylate inducer as well as giving insights into the signal propagation mechanism in LysR-type transcription factors (paper I). DntR mutant crystal structures combined with the modelling of DntR-2,4-dnt interactions led to the design of a DntR mutant that has a limited response to 2,4-dnt in a whole cell biosensor system (paper 2). Crystal structures of apo-NikR from H. pylori (HpNikR) and of Ni-bound intermediary states of the protein were obtained. The latter have helped in unravelling the Ni incorporation and selectivity mechanisms of NikRs and have shown a strong cooperativity between conformational changes in the Ni binding domain with movements of the DNA binding domain (paper 3). Biochemical studies and comparisons of the HpNikR crystal structures with those of NikR homologues strongly suggest that HpNikR has evolved different surface properties (paper 4) and a new mode of DNA binding.

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  • 40. Dzintars, Eric
    et al.
    Stathakis, Sotirios
    Mavroidis, Panayiotis
    Stockholm University, Faculty of Science, Department of Physics.
    Sadeghi, Amir
    Papanikolaou, Nikos
    Performance of independent dose calculation in helical tomotherapy: implementation of the mcsim code2012In: Australasian Physical & Engineering Sciences in Medicine, ISSN 0158-9938, Vol. 35, no 4, p. 423-438Article in journal (Refereed)
    Abstract [en]

    Currently, a software-based second check dose calculation for helical tomotherapy (HT) is not available. The goal of this study is to evaluate the dose calculation accuracy of the in-house software using EGS4/MCSIM Monte Carlo environment against the treatment planning system calculations. In-house software was used to convert HT treatment plan information into a non-helical format. The MCSIM dose calculation code was evaluated by comparing point dose calculations and dose profiles against those from the HT treatment plan. Fifteen patients, representing five treatment sites, were used in this comparison. Point dose calculations between the HT treatment planning system and the EGS4/MCSIM Monte Carlo environment had percent difference values below 5 % for the majority of this study. Vertical and horizontal planar profiles also had percent difference values below 5 % for the majority of this study. Down sampling was seen to improve speed without much loss of accuracy. EGS4/MCSIM Monte Carlo environment showed good agreement with point dose measurements, compared to the HT treatment plans. Vertical and horizontal profiles also showed good agreement. Significant time saving may be obtained by down-sampling beam projections. The dose calculation accuracy of the in-house software using the MCSIM code against the treatment planning system calculations was evaluated. By comparing point doses and dose profiles, the EGS4/MCSIM Monte Carlo environment was seen to provide an accurate independent dose calculation.

  • 41. Edger, Patrick P.
    et al.
    Heidel-Fischer, Hanna M.
    Bekaert, Michael
    Rota, Jadranka
    Gloeckner, Gernot
    Platts, Adrian E.
    Heckel, David G.
    Der, Joshua P.
    Wafula, Eric K.
    Tang, Michelle
    Hofberger, Johannes A.
    Smithson, Ann
    Hall, Jocelyn C.
    Blanchette, Matthieu
    Bureau, Thomas E.
    Wright, Stephen I.
    dePamphilis, Claude W.
    Schranz, M. Eric
    Barker, Michael S.
    Conant, Gavin C.
    Wahlberg, Niklas
    Vogel, Heiko
    Pires, J. Chris
    Wheat, Christopher W.
    Stockholm University, Faculty of Science, Department of Zoology.
    The butterfly plant arms-race escalated by gene and genome duplications2015In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 112, no 27, p. 8362-8366Article in journal (Refereed)
    Abstract [en]

    Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.

  • 42.
    Eriksson, Olaspers Sara
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Geörg, Miriam
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sjölinder, Hong
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sillard, Rannar
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Lindberg, Staffan
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Jonsson, Ann-Beth
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Identification of Cell-Penetrating Peptides That Are Bactericidal to Neisseria meningitidis and Prevent Inflammatory Responses upon Infection2013In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 57, no 8, p. 3704-3712Article in journal (Refereed)
    Abstract [en]

    Meningococcal disease is characterized by a fast progression and a high mortality rate. Cell-penetrating peptides (CPPs), developed as vectors for cargo delivery into eukaryotic cells, share structural features with antimicrobial peptides. A screen identified two CPPs, transportan-10 (TP10) and model amphipathic peptide (MAP), with bactericidal action against Neisseria meningitidis. Both peptides were active in human whole blood at micromolar concentrations, while hemolysis remained negligible. Additionally, TP10 exhibited significant antibacterial activity in vivo. Uptake of SYTOX green into live meningococci was observed within minutes after TP10 treatment, suggesting that TP10 may act by membrane permeabilization. Apart from its bactericidal activity, TP10 suppressed inflammatory cytokine release from macrophages infected with N. meningitidis as well as from macrophages stimulated with enterobacterial and meningococcal lipopolysaccharide (LPS). Finally, incubation with TP10 reduced the binding of LPS to macrophages. This novel endotoxin-inhibiting property of TP10, together with its antimicrobial activity in vivo, indicates the possibility to design peptide-based therapies for infectious diseases.

  • 43.
    Eriksson, Olivia
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Brinne, Björn
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhou, Yishao
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Björkegren, Johan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tegnér, Jesper
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Deconstructing the core dynamics from a complex time-lagged regulatory biological circuit2009In: IET systems biology, ISSN 1751-8849, Vol. 3, no 2, p. 113-129Article in journal (Refereed)
    Abstract [en]

    Complex regulatory dynamics is ubiquitous in molecular networks composed of genes and proteins. Recent progress in computational biology and its application to molecular data generate a growing number of complex networks. Yet, it has been difficult to understand the governing principles of these networks beyond graphical analysis or extensive numerical simulations. Here the authors exploit several simplifying biological circumstances which thereby enable to directly detect the underlying dynamical regularities driving periodic oscillations in a dynamical nonlinear computational model of a protein-protein network. System analysis is performed using the cell cycle, a mathematically well-described complex regulatory circuit driven by external signals. By introducing an explicit time delay and using a -tearing-and-zooming- approach the authors reduce the system to a piecewise linear system with two variables that capture the dynamics of this complex network. A key step in the analysis is the identification of functional subsystems by identifying the relations between state-variables within the model. These functional subsystems are referred to as dynamical modules operating as sensitive switches in the original complex model. By using reduced mathematical representations of the subsystems the authors derive explicit conditions on how the cell cycle dynamics depends on system parameters, and can, for the first time, analyse and prove global conditions for system stability. The approach which includes utilising biological simplifying conditions, identification of dynamical modules and mathematical reduction of the model complexity may be applicable to other well-characterised biological regulatory circuits.

  • 44.
    Esbjörner, Elin K.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Oglęcka, Kamila
    Lincoln, Per
    Gräslund, Astrid
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nordén, Bengt
    Membrane binding of pH-sensitive Influenza fusion peptides. Positioning, configuration and induced leakage in lipid vesicles models2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 47, p. 13490-13504Article in journal (Refereed)
    Abstract [en]

    pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA 1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion (∼60-65° relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue

  • 45.
    Ezzat, Kariem
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides (CPPs) that have superior delivery properties for several nucleic acid-based therapeutics are developed. These CPPs can spontaneously form nanoparticles upon non-covalent complexation with the nucleic acid cargo, and the formed nanoparticles mediate efficient cellular transfection. In paper I, we show that an N-terminally stearic acid-modified version of transportan-10 (PF3) can efficiently transfect different cell types with plasmid DNA and mediates efficient gene delivery in-vivo when administrated intra muscularly (i.m.) or intradermaly (i.d.). In paper II, a new peptide with ornithine modification, PF14, is shown to efficiently deliver splice-switching oligonucleotides (SSOs) in different cell models including mdx mouse myotubes; a cell culture model of Duchenne’s muscular dystrophy (DMD). Additionally, we describe a method for incorporating the PF14-SSO nanoparticles into a solid formulation that is active and stable even when stored at elevated temperatures for several weeks. In paper III, we demonstrate the involvement of class-A scavenger receptor subtypes (SCARA3 & SCARA5) in the uptake of PF14-SSO nanoparticles, which possess negative surface charge, and suggest for the first time that some CPP-based systems function through scavenger receptors. In paper IV, the ability of PF14 to deliver siRNA to different cell lines is shown and their stability in simulated gastric acidic conditions is highlighted.

    Taken together, these results demonstrate that certain chemical modifications can drastically enhance the activity and stability of CPPs for delivering nucleic acids after spontaneous nanoparticle formation upon non-covalent complexation. Moreover, we show that CPP-based nanoparticles can be formulated into convenient and stable solid formulations that can be suitable for several therapeutic applications. Importantly, the involvement of scavenger receptors in the uptake of such nanoparticles is presented, which could yield novel possibilities to understand and improve the transfection by CPPs and other gene therapy nanoparticles.

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  • 46.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. Karolinska Institutet, Sweden.
    Pernemalm, Maria
    Pålsson, Sandra
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Roberts, Thomas C.
    Järver, Peter
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Dondalska, Aleksandra
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Bestas, Burcu
    Sobkowiak, Michal J.
    Levanen, Bettina
    Skold, Magnus
    Thompson, Elizabeth A.
    Saher, Osama
    Kari, Otto K.
    Lajunen, Tatu
    Ekström, Eva Sverremark
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Nilsson, Caroline
    Ishchenko, Yevheniia
    Malm, Tarja
    Wood, Matthew J. A.
    Power, Ultan F.
    Masich, Sergej
    Linden, Anders
    Sandberg, Johan K.
    Lehtio, Janne
    Spetz, Anna-Lena
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    EL Andaloussi, Samir
    The viral protein corona directs viral pathogenesis and amyloid aggregation2019In: Nature Communications, E-ISSN 2041-1723, Vol. 10, article id 2331Article in journal (Refereed)
    Abstract [en]

    Artificial nanoparticles accumulate a protein corona layer in biological fluids, which significantly influences their bioactivity. As nanosized obligate intracellular parasites, viruses share many biophysical properties with artificial nanoparticles in extracellular environments and here we show that respiratory syncytial virus (RSV) and herpes simplex virus type 1 (HSV-1) accumulate a rich and distinctive protein corona in different biological fluids. Moreover, we show that corona pre-coating differentially affects viral infectivity and immune cell activation. In addition, we demonstrate that viruses bind amyloidogenic peptides in their corona and catalyze amyloid formation via surface-assisted heterogeneous nucleation. Importantly, we show that HSV-1 catalyzes the aggregation of the amyloid beta-peptide (A beta(42)), a major constituent of amyloid plaques in Alzheimer's disease, in vitro and in animal models. Our results highlight the viral protein corona as an acquired structural layer that is critical for viral-host interactions and illustrate a mechanistic convergence between viral and amyloid pathologies.

  • 47.
    Ezzat, Kariem
    et al.
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Zaghloul, Eman M.
    EL Andaloussi, Samir
    Lehto, Taavi
    Hilal, Ramy
    Magdy, Tarek
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Smith, Edvard C. I.
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry.
    Solid formulation of cell-penetrating peptide nanoparticles with siRNA and their stability in simulated gastric conditions2012In: Journal of Controlled Release, ISSN 0168-3659, E-ISSN 1873-4995, Vol. 162, no 1, p. 1-8Article in journal (Refereed)
    Abstract [en]

    Cell-penetrating peptides (CPPs) are short cationic peptides that have been extensively studied as drug delivery vehicles for proteins, nucleic acids and nanoparticles. However, the formulation of CPP-based therapeutics into different pharmaceutical formulations and their stability in relevant biological environments have not been given the same attention. Here, we show that a newly developed CPP, PepFect 14 (PF14), forms non-covalent nanocomplexes with short interfering RNA (siRNA), which are able to elicit efficient RNA-interference (RNAi) response in different cell-lines. RNAi effect was obtained at low siRNA doses with a unique kinetic profile. Furthermore, we utilized the solid dispersion technique to formulate PF14/siRNA nanocomplexes into solid formulations that were as active as the freshly prepared nanocomplexes in solution. Importantly, the freshly prepared nanocomplexes and solid formulations were stable after incubation with simulated gastric fluid having a pH of 1.2 and containing proteolytic enzymes. These results demonstrate the activity of PF14 in delivering and protecting siRNA in different pharmaceutical forms and biological environments.

  • 48. Faxén, Kristina
    et al.
    Brzezinski, Peter
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase.2007In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1767, no 5, p. 381-386Article in journal (Refereed)
    Abstract [en]

    Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).

  • 49. Ferreira, Brigida C.
    et al.
    Lopes, Maria do Carmo
    Mateus, Josefina
    Capela, Miguel
    Mavroidis, Panayiotis
    Stockholm University, Faculty of Science, Medical Radiation Physics (together with KI).
    Radiobiological evaluation of forward and inverse IMRT using different fractionations for head and neck tumours2010In: Radiation Oncology, E-ISSN 1748-717X, Vol. 5, p. 57-Article in journal (Refereed)
    Abstract [en]

    Purpose: To quantify the radiobiological advantages obtained by an Improved Forward Planning technique (IFP) and two IMRT techniques using different fractionation schemes for the irradiation of head and neck tumours. The conventional radiation therapy technique (CONVT) was used here as a benchmark. Methods: Seven patients with head and neck tumours were selected for this retrospective planning study. The PTV1 included the primary tumour, PTV2 the high risk lymph nodes and PTV3 the low risk lymph nodes. Except for the conventional technique where a maximum dose of 64.8 Gy was prescribed to the PTV1, 70.2 Gy, 59.4 Gy and 50.4 Gy were prescribed respectively to PTV1, PTV2 and PTV3. Except for IMRT2, all techniques were delivered by three sequential phases. The IFP technique used five to seven directions with a total of 15 to 21 beams. The IMRT techniques used five to nine directions and around 80 segments. The first, IMRT1, was prescribed with the conventional fractionation scheme of 1.8 Gy per fraction delivered in 39 fractions by three treatment phases. The second, IMRT2, simultaneously irradiated the PTV2 and PTV3 with 59.4 Gy and 50.4 Gy in 28 fractions, respectively, while the PTV1 was boosted with six subsequent fractions of 1.8 Gy. Tissue response was calculated using the relative seriality model and the Poisson Linear-Quadratic-Time model to simulate repopulation in the primary tumour. Results: The average probability of total tumour control increased from 38% with CONVT to 80% with IFP, to 85% with IMRT1 and 89% with IMRT2. The shorter treatment time and larger dose per fraction obtained with IMRT2 resulted in an 11% increase in the probability of control in the PTV1 with respect to IFP and 7% relatively to IMRT1 (p < 0.05). The average probability of total patient complications was reduced from 80% with CONVT to 61% with IFP and 31% with IMRT. The corresponding probability of complications in the ipsilateral parotid was 63%, 42% and 20%; in the contralateral parotid it was 50%, 20% and 9%; in the oral cavity it was 2%, 15% and 4% and in the mandible it was 1%, 5% and 3%, respectively. Conclusions: A significant improvement in treatment outcome was obtained with IMRT compared to conventional radiation therapy. The practical and biological advantages of IMRT2, employing a shorter treatment time, may outweigh the small differences obtained in the organs at risk between the two IMRT techniques. This technique is therefore presently being used in the clinic for selected patients with head and neck tumours. A significant improvement in the quality of the dose distribution was obtained with IFP compared to CONVT. Thus, this beam arrangement is used in the clinical routine as an alternative to IMRT.

  • 50. Freimann, Krista
    et al.
    Arukuusk, Piret
    Kurrikoff, Kaido
    Pärnaste, Ly
    Raid, Raivo
    Piirsoo, Andres
    Pooga, Margus
    Langel, Ülo
    Stockholm University, Faculty of Science, Department of Neurochemistry. University of Tartu, Estonia.
    Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo2018In: Molecular Therapy Nucleic Acids, E-ISSN 2162-2531, Vol. 10, p. 28-35Article in journal (Refereed)
    Abstract [en]

    Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.

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