Browsing by Subject "IN-VIVO"

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  • Moilanen, Anne-Mari; Rysa, Jaana; Serpi, Raisa; Mustonen, Erja; Szabo, Zoltan; Aro, Jani; Napankangas, Juha; Tenhunen, Olli; Sutinen, Meeri; Salo, Tuula; Ruskoaho, Heikki (2012)
  • Zhou, Qi-Hang; Qin, Wei-Wei; Finel, Moshe; He, Qing-Qing; Tu, Dong-Zhu; Wang, Chao-Ran; Ge, Guang-Bo (2021)
    Strong inhibition of the human UDP-glucuronosyltransferase enzymes (UGTs) may lead to undesirable effects, including hyperbilirubinaemia and drugiherb-drug interactions. Currently, there is no good way to examine the inhibitory effects and specificities of compounds toward all the important human UGTs, side-by-side and under identical conditions. Herein, we report a new, broad-spectrum substrate for human UGTs and its uses in screening and characterizing of UGT inhibitors. Following screening a variety of phenolic compound(s), we have found that methylophiopogonanone A (MOA) can be readily O-glucuronidated by all tested human UGTs, including the typical N-glucuronidating enzymes UGT1A4 and UGT2B10. MOA-O-glucuronidation yielded a single mono-O-glucuronide that was biosynthesized and purified for structural characterization and for constructing an LC-UV based MOA-O-glucuronidation activity assay, which was then used for investigating MOA-O-glucuronidation kinetics in recombinant human UGTs. The derived K-m values were crucial for selecting the most suitable assay conditions for assessing inhibitory potentials and specificity of test compound(s). Furthermore, the inhibitory effects and specificities of four known UGT inhibitors were reinvestigated by using MOA as the substrate for all tested UGTs. Collectively, MOA is a broad-spectrum substrate for the human UGTs, which offers a new and practical tool for assessing inhibitory effects and specificities of UGT inhibitors. (C) 2021 Elsevier B.V. All rights reserved.
  • Haegerling, Rene; Pollmann, Cathrin; Andreas, Martin; Schmidt, Christian; Nurmi, Harri; Adams, Ralf H.; Alitalo, Kari; Andresen, Volker; Schulte-Merker, Stefan; Kiefer, Friedemann (2013)
  • Lewis, Samantha C.; Joers, Priit; Willcox, Smaranda; Griffith, Jack D.; Jacobs, Howard T.; Hyman, Bradley C. (2015)
    Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
  • Ma, Cheng; Hao, Zhenyu; Huysmans, Gerard; Lesiuk, Amelia; Bullough, Per; Wang, Yingying; Bartlam, Mark; Phillips, Simon E.; Young, James D.; Goldman, Adrian; Baldwin, Stephen A.; Postis, Vincent L. G. (2015)
    Membrane proteins play key roles in many biological processes, from acquisition of nutrients to neurotransmission, and are targets for more than 50% of current therapeutic drugs. However, their investigation is hampered by difficulties in their production and purification on a scale suitable for structural studies. In particular, the nature and location of affinity tags introduced for the purification of recombinant membrane proteins can greatly influence their expression levels by affecting their membrane insertion. The extent of such effects typically depends on the transmembrane topologies of the proteins, which for proteins of unknown structure are usually uncertain. For example, attachment of oligohistidine tags to the periplasmic termini of membrane proteins often interferes with folding and drastically impairs expression in Escherichia coli. To circumvent this problem we have employed a novel strategy to enable the rapid production of constructs bearing a range of different affinity tags compatible with either cytoplasmic or periplasmic attachment. Tags include conventional oligohistidine tags compatible with cytoplasmic attachment and, for attachment to proteins with a periplasmic terminus, either tandem Strep-tag II sequences or oligohistidine tags fused to maltose binding protein and a signal sequence. Inclusion of cleavage sites for TEV or HRV-3C protease enables tag removal prior to crystallisation trials or a second step of purification. Together with the use of bioinformatic approaches to identify members of membrane protein families with topologies favourable to cytoplasmic tagging, this has enabled us to express and purify multiple bacterial membrane transporters. To illustrate this strategy, we describe here its use to purify bacterial homologues of human membrane proteins from the Nramp and ZIP families of divalent metal cation transporters and from the concentrative nucleoside transporter family. The proteins are expressed in E. coli in a correctly folded, functional state and can be purified in amounts suitable for structural investigations.
  • Kopasz, Anna Georgina; Pusztai, David Zsolt; Karkas, Reka; Hudoba, Liza; Abdullah, Khaldoon Sadiq Ahmed; Imre, Gergely; Pankotai-Bodo, Gabriella; Migh, Ede; Nagy, Andrea; Kriston, Andras; German, Peter; Drubi, Andrea Bakne; Molnar, Anna; Fekete, Ildiko; Dani, Virag Eva; Ocsovszki, Imre; Puskas, Laszlo Geza; Horvath, Peter; Sukosd, Farkas; Mates, Lajos (2022)
    Background Understanding the contribution of gene function in distinct organ systems to the pathogenesis of human diseases in biomedical research requires modifying gene expression through the generation of gain- and loss-of-function phenotypes in model organisms, for instance, the mouse. However, methods to modify both germline and somatic genomes have important limitations that prevent easy, strong, and stable expression of transgenes. For instance, while the liver is remarkably easy to target, nucleic acids introduced to modify the genome of hepatocytes are rapidly lost, or the transgene expression they mediate becomes inhibited due to the action of effector pathways for the elimination of exogenous DNA. Novel methods are required to overcome these challenges, and here we develop a somatic gene delivery technology enabling long-lasting high-level transgene expression in the entire hepatocyte population of mice. Results We exploit the fumarylacetoacetate hydrolase (Fah) gene correction-induced regeneration in Fah-deficient livers, to demonstrate that such approach stabilizes luciferase expression more than 5000-fold above the level detected in WT animals, following plasmid DNA introduction complemented by transposon-mediated chromosomal gene transfer. Building on this advancement, we created a versatile technology platform for performing gene function analysis in vivo in the mouse liver. Our technology allows the tag-free expression of proteins of interest and silencing of any arbitrary gene in the mouse genome. This was achieved by applying the HADHA/B endogenous bidirectional promoter capable of driving well-balanced bidirectional expression and by optimizing in vivo intronic artificial microRNA-based gene silencing. We demonstrated the particular usefulness of the technology in cancer research by creating a p53-silenced and hRas G12V-overexpressing tumor model. Conclusions We developed a versatile technology platform for in vivo somatic genome editing in the mouse liver, which meets multiple requirements for long-lasting high-level transgene expression. We believe that this technology will contribute to the development of a more accurate new generation of tools for gene function analysis in mice.
  • Räisänen, Ismo T.; Sorsa, Timo; van der Schoor, Gerrit-Jan; Tervahartiala, Taina; van der Schoor, Peter; Gieselmann, Dirk-Rolf; Heikkinen, Anna Maria (2019)
    This cross-sectional study compares the effectiveness of an active MMP-8 (aMMP-8) point-of-care (PoC)/chairside mouthrinse test to the conventional bleeding on probing (BOP) (cutoff 20%) test in detecting subclinical periodontitis/pre-periodontitis in Finnish adolescents. The study was carried out at the Kotka Health Center, Finland. A total of 47 adolescents (30 boys/17 girls) aged 15-17 were first tested with the aMMP-8 PoC test, followed by a full-mouth evaluation of clinical parameters of oral health including periodontal, oral mucosal, and caries assessment. A periodontist performed these clinical examinations. The aMMP-8 PoC test result had much stronger association with subclinical periodontitis than the BOP 20% test (2.8-5.3 times stronger in terms of odds ratio). The aMMP-8 PoC test had >= 2 times higher sensitivity than the BOP 20% test with, generally, the same specificity. Further, the aMMP-8 PoC test had generally better accuracy and lower false negative percentages. The aMMP-8 PoC test seemed to be more effective than the conventional BOP test in detecting subclinical periodontitis/pre-periodontitis in adolescents reducing the risk of their undertreatment. However, the sample size may be a limiting factor, and more studies are needed to confirm our results for both adolescents and adults.
  • Vashchinkina, Elena; Piippo, Ossi; Vekovischeva, Olga; Krupitsky, Evgeny; Ilyuk, Ruslan; Neznanov, Nikholay; Kazankov, Kirill; Zaplatkin, Igor; Korpi, Esa R. (2018)
    The gabapentinoid pregabalin is a rapid-acting anxiolytic and analgesic, possibly suitable in supervised opioid detoxification. However, clinicians have been cautious in using it because of its unknown addictive risk and rising number of mortalities after pregabalin self-medication in opioid abusers. Here, we studied interactions of pregabalin and morphine on reward functions of the dopamine system in mice and the efficacy of pregabalin on withdrawal in opioid addicts. After the treatment of mice with pregabalin and morphine, we used electrophysiology to study neuroplasticity in midbrain slices, self-administration and conditioned place preference tests to investigate the rewarding potential of pregabalin and naloxone-precipitated morphine withdrawal to evaluate opioid withdrawal symptoms. Further, we ran a pilot single-blind, randomized, controlled trial (34 heroin addicts) to evaluate the efficacy and safety of pregabalin in the treatment of opioid withdrawal syndrome. Pregabalin alone did not induce glutamate receptor neuroplasticity of dopamine neurons in the ventral tegmental area, but pre-treatment with pregabalin suppressed morphine-induced neuroplasticity, hyperlocomotion and morphine self-administration. Pregabalin administration after chronic morphine exposure failed to induce any rewarding effects. Instead, pregabalin suppressed withdrawal symptoms in both morphine-treated mice and opioid addicts and was well tolerated. Intriguingly, pregabalin administration after a low dose of morphine strongly facilitated ventral tegmental area neuroplasticity and led to increased conditioned place preference. Pregabalin appears to have the efficacy to counteract both reinforcing and withdrawal effects of opioids, but it also has a potentiating effect when given to mice with existing opioid levels.
  • Rautaniemi, Kaisa; Zini, Jacopo; Löfman, Emilia; Saari, Heikki; Haapalehto, Iida; Laukka, Johanna; Vesamäki, Sami; Efimov, Alexander; Yliperttula, Marjo; Laaksonen, Timo; Vuorimaa-Laukkanen, Elina; Lisitsyna, Ekaterina S. (2022)
    Studies of extracellular vesicles (EVs), their trafficking and characterization often employ fluorescent labelling. Unfortunately, little attention has been paid thus far to a thorough evaluation of the purification of EVs after labelling, although the presence of an unbound dye may severely compromise the results or even lead to wrong conclusions on EV functionality. Here, we systematically studied five dyes for passive EV labelling and meticulously compared five typical purification methods: ultracentrifugation (UC), ultracentrifugation with discontinuous density gradient (UCG), ultrafiltration (UF), size exclusion chromatography (SEC), and anion exchange chromatography (AEC). A general methodology for evaluation of EV purification efficiency after the labelling was developed and tested to select the purification methods for the chosen dyes. Firstly, we found that some methods initially lead to high EV losses even in the absence of the dye. Secondly, the suitable purification method needs to be found for each particular dye and depends on the physical and chemical properties of the dye. Thirdly, we demonstrated that the developed parameter E-rp (relative purification efficiency) is a useful tool for the pre-screening of the suitable dye-purification method combinations. Additionally, it was also shown that the labelled EVs properly purified from the unbound dye may show significantly reduced contrast and visibility in the target application, e.g. in the live cell fluorescence lifetime imaging.
  • Rademakers, Timo; van der Vorst, Emiel P. C.; Daissormont, Isabelle T. M. N.; Otten, Jeroen J. T.; Theodorou, Kosta; Theelen, Thomas L.; Gijbels, Marion; Anisimov, Andrey; Nurmi, Harri; Lindeman, Jan H. N.; Schober, Andreas; Heeneman, Sylvia; Alitalo, Kari; Biessen, Erik A. L. (2017)
    During plaque progression, inflammatory cells progressively accumulate in the adventitia, paralleled by an increased presence of leaky vasa vasorum. We here show that next to vasa vasorum, also the adventitial lymphatic capillary bed is expanding during plaque development in humans and mouse models of atherosclerosis. Furthermore, we investigated the role of lymphatics in atherosclerosis progression. Dissection of plaque draining lymph node and lymphatic vessel in atherosclerotic ApoE(-/-)mice aggravated plaque formation, which was accompanied by increased intimal and adventitial CD3(+) T cell numbers. Likewise, inhibition of VEGF-C/D dependent lymphangiogenesis by AAV aided gene transfer of hVEGFR3-Ig fusion protein resulted in CD3(+) T cell enrichment in plaque intima and adventitia. hVEGFR3-Ig gene transfer did not compromise adventitial lymphatic density, pointing to VEGF-C/D independent lymphangiogenesis. We were able to identify the CXCL12/CXCR4 axis, which has previously been shown to indirectly activate VEGFR3, as a likely pathway, in that its focal silencing attenuated lymphangiogenesis and augmented T cell presence. Taken together, our study not only shows profound, partly CXCL12/CXCR4 mediated, expansion of lymph capillaries in the adventitia of atherosclerotic plaque in humans and mice, but also is the first to attribute an important role of lymphatics in plaque T cell accumulation and development.
  • El-Khoury, Riyad; Dufour, Eric; Rak, Malgorzata; Ramanantsoa, Nelina; Grandchamp, Nicolas; Csaba, Zsolt; Duvillie, Bertrand; Benit, Paule; Gallego, Jorge; Gressens, Pierre; Sarkis, Chamsy; Jacobs, Howard T.; Rustin, Pierre (2013)
  • V. Almeida, Patrick; Shahbazi, Mohammad-Ali; Mäkilä, Ermei; Kaasalainen, Martti; Salonen, Jarno; Hirvonen, Jouni; Santos, Helder A. (2014)
  • V. Almeida, Patrick; Shahbazi, Mohammad-Ali; Mäkilä, Ermei; Kaasalainen, Martti; Salonen, Jarno; Hirvonen, Jouni; Santos, Helder A. (The Royal Society of Chemistry, 2014)
  • Georgiadou, Maria; Lilja, Johanna; Jacquemet, Guillaume; Guzman, Camilo; Rafaeva, Maria; Alibert, Charlotte; Yan, Yan; Sahgal, Pranshu; Lerche, Martina; Manneville, Jean-Baptiste; Makela, Tomi P.; Ivaska, Johanna (2017)
    Tight regulation of integrin activity is paramount for dynamic cellular functions such as cell matrix adhesion and mechanotransduction. Integrin activation is achieved through intracellular interactions at the integrin cytoplasmic tails and through integrin-ligand binding. In this study, we identify the metabolic sensor AMP-activated protein kinase (AMPK) as a beta 1-integrin inhibitor in fibroblasts. Loss of AMPK promotes beta 1-integrin activity, the formation of centrally located active beta 1-integrin- and tensin-rich mature fibrillar adhesions, and cell spreading. Moreover, in the absence of AMPK, cells generate more mechanical stress and increase fibronectin fibrillogenesis. Mechanistically, we show that AMPK negatively regulates the expression of the integrin-binding proteins tensin1 and tensin3. Transient expression of tensins increases beta 1-integrin activity, whereas tensin silencing reduces integrin activity in fibroblasts lacking AMPK. Accordingly, tensin silencing in AMPK-depleted fibroblasts impedes enhanced cell spreading, traction stress, and fibronectin fiber formation. Collectively, we show that the loss of AMPK up-regulates tensins, which bind beta 1-integrins, supporting their activity and promoting fibrillar adhesion formation and integrin-dependent processes.
  • Ni, Ruiqing; Gillberg, Per-Goran; Bogdanovic, Nenad; Viitanen, Matti; Myllykangas, Liisa; Nennesmo, Inger; Langstrom, Bengt; Nordberg, Agneta (2017)
    Introduction: Amyloid imaging has been integrated into diagnostic criteria for Alzheimer's disease (AD). How amyloid tracers binding differ for different tracer structures and amyloid-beta aggregates in autosomal dominant AD (ADAD) and sporadic AD is unclear. Methods: Binding properties of different amyloid tracers were examined in brain homogenates from six ADAD with APPswe, PS1 M146V, and PS1 E Delta 9 mutations, 13 sporadic AD, and 14 control cases. Results: H-3-PIB, H-3-florbetaben, H-3-AZD2184, and BTA-1 shared a high-and a varying low-affinity binding site in the frontal cortex of sporadic AD. AZD2184 detected another binding site (affinity 33 nM) in the frontal cortex of ADAD. The H-3-AZD2184 and H-3-PIB binding were significantly higher in the striatum of ADAD compared to sporadic AD and control. Polyphenol resveratrol showed strongest inhibition on H-3-AZD84 binding followed by H-3-florbetaben and minimal on H-3-PIB. Discussion: This study implies amyloid tracers of different structures detect different sites on amyloid-beta fibrils or conformations. (C) 2016 the Alzheimer's Association. Published by Elsevier Inc. All rights reserved.
  • Simmons, Suzanne C.; Jaemsae, Hannaleena; Silva, Dilson; Cortez, Celia M.; McKenzie, Edward A.; Bitu, Carolina C.; Salo, Sirpa; Nurmenniemi, Sini; Nyberg, Pia; Risteli, Juha; deAlmeida, Carlos E. B.; Brenchley, Paul E. C.; Salo, Tuula; Missailidisi, Sotiris (2014)
  • Knuuttila, Matias; Mehmood, Arfa; Huhtaniemi, Riikka; Yatkin, Emrah; Häkkinen, Merja R.; Oksala, Riikka; Laajala, Teemu D.; Ryberg, Henrik; Handelsman, David J.; Aittokallio, Tero; Auriola, Seppo; Ohlsson, Claes; Laiho, Asta; Elo, Laura L.; Sipila, Petra; Makela, Sari I.; Poutanen, Matti (2018)
    The development of castration-resistant prostate cancer (CRPC) is associated with the activation of intratumoral androgen biosynthesis and an increase in androgen receptor (AR) expression. We recently demonstrated that, similarly to the clinical CRPC, orthotopically grown castration-resistant VCaP (CR-VCaP) xenografts express high levels of AR and retain intratumoral androgen concentrations similar to tumors grown in intact mice. Herein, we show that antiandrogen treatment (enzalutamide or ARN-509) significantly reduced (10-fold, P <0.01) intratumoral testosterone and dihydrotestosterone concentrations in the CR-VCaP tumors, indicating that the reduction in intratumoral androgens is a novel mechanism by which antiandrogens mediate their effects in CRPC. Antiandrogen treatment also altered the expression of multiple enzymes potentially involved in steroid metabolism. Identical to clinical CRPC, the expression levels of the full-length AR (twofold, P <0.05) and the AR splice variants 1 (threefold, P <0.05) and 7 (threefold, P <0.01) were further increased in the antiandrogen-treated tumors. Nonsignificant effects were observed in the expression of certain classic androgen-regulated genes, such as TMPRSS2 and KLK3, despite the low levels of testosterone and dihydrotestosterone. However, other genes recently identified to be highly sensitive to androgen-regulated AR action, such as NOV and ST6GalNAc1, were markedly altered, which indicated reduced androgen action. Taken together, the data indicate that, besides blocking AR, antiandrogens modify androgen signaling in CR-VCaP xenografts at multiple levels.
  • Perjes, Abel; Skoumal, Reka; Tenhunen, Olli; Konyi, Attila; Simon, Mihaly; Horvath, Ivan G.; Kerkelä, Risto; Ruskoaho, Heikki; Szokodi, Istvan (2014)
  • Fusciello, Manlio; Fontana, Flavia; Tähtinen, Siri; Capasso, Cristian; Feola, Sara; da Silva Lopes Martins, Beatriz; Chiaro, Jacopo; Peltonen, Karita; Ylösmäki, Leena; Ylösmäki, Erkko; Hamdan Hissaoui, Firas; Kari, Otto K.; Ndika, Joseph; Alenius, Harri; Urtti, Arto; Hirvonen, Jouni T.; Santos, Hélder A.; Cerullo, Vincenzo (2019)
    Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.
  • Lindgren, Noora; Tuisku, Jouni; Vuoksimaa, Eero; Helin, Semi; Karrasch, Mira; Marjamäki, Päivi; Kaprio, Jaakko; Rinne, Juha O. (2020)
    Alzheimer's disease is associated with chronic response of innate immune system, referred as neuroinflammation. PET radioligands binding to the 18kDa translocator protein are potential biomarkers of neuroinflammation. Translocator protein PET studies in mild cognitive impairment and Alzheimer's disease have indicated controversial results, possibly reflecting interindividual variation and heterogeneity of study populations. We controlled for genetic and environmental effects by studying twin pairs discordant for episodic memory performance. Episodic memory impairment is a well-known cognitive hallmark of early Alzheimer's disease process. Eleven same-sex twin pairs (four monozygotic pairs, six female pairs, age 72-77 years) underwent [C-11]N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine ([C-11]PBR28) PET imaging, structural magnetic resonance imaging and neuropsychological testing in 2014-17. Main PET outcome was the volume-weighted average standardized uptake value of cortical regions vulnerable to Alzheimer's disease pathology. Ten pairs were discordant for episodic memory performance. In the eight pairs with identical translocator protein genotype, twins with poorer episodic memory had similar to 20% higher cortical [C-11]PBR28 binding compared with their better-performing co-twins (mean intra-pair difference 0.21 standardized uptake value, 95% confidence interval 0.05-0.37, P = 0.017). The result remained the same when including all discordant pairs and controlling for translocator protein genotype. Increased translocator protein PET signal suggests that increased microglial activation is associated with poorer episodic memory performance. Twins with worse episodic memory performance compared with their co-twins had on average 20% higher uptake of the neuroinflammatory marker translocator protein PET tracer (11)[C-11]PBR28. The findings support a negative association between neuroinflammation and episodic memory and the use of translocator protein positron emission tomography as a useful indicator of Alzheimer's disease process.