Browsing by Subject "PROBES"

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  • Wang, Linping; Saarela, Jani; Poque, Sylvain; Valkonen, Jari P. T. (2020)
    The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.
  • Tang, Jing; Tanoli, Zia-ur-Rehman; Ravikumar, Balaguru; Alam, Zaid; Rebane, Anni; Vähä-Koskela, Markus; Peddinti, Gopal; van Adrichem, Arjan J.; Wakkinen, Janica; Jaiswal, Alok; Karjalainen, Ella; Gautam, Prson; He, Liye; Parri, Elina; Khan, Suleiman; Gupta, Abhishekh; Ali, Mehreen; Yetukuri, Laxman; Gustavsson, Anna-Lena; Seashore-Ludlow, Brinton; Hersey, Anne; Leach, Andrew R.; Overington, John P.; Repasky, Gretchen; Wennerberg, Krister; Aittokallio, Tero (2018)
    Knowledge of the full target space of bioactive substances, approved and investigational drugs as well as chemical probes, provides important insights into therapeutic potential and possible adverse effects. The existing compound-target bioactivity data resources are often incomparable due to non-standardized and heterogeneous assay types and variability in endpoint measurements. To extract higher value from the existing and future compound target-profiling data, we implemented an open-data web platform, named Drug Target Commons (DTC), which features tools for crowd-sourced compound-target bioactivity data annotation, standardization, curation, and intra-resource integration. We demonstrate the unique value of DTC with several examples related to both drug discovery and drug repurposing applications and invite researchers to join this community effort to increase the reuse and extension of compound bioactivity data.
  • Kärnä-Behm, Jaana (2019)
    The purpose of this study is to promote the experiential learning (EL) method in the pedagogics of art and design in higher education. This article is based on a case study consisting of two pedagogical projects in interior design courses, the probing project and the multisensory space project, carried out between 2014 and 2016 with trainee teachers. Using the data from these projects I analyse using the qualitative content analysis method how and with what implications EL supports learning of art and design in higher education. The results show that EL was found to be inspiring and self-expressive, and was an unusual and motivating way to learn interior design. In a teacher education context EL gave students ideas about collaborative and EL-based methods of learning that could be applied to their own future teaching projects.
  • Aaltonen, Niina; Singha, Prosanta K.; Jakupovic, Hermina; Wirth, Thomas; Samaranayake, Haritha; Pasonen-Seppanen, Sanna; Rilla, Kirsi; Varjosalo, Markku; Edgington-Mitchell, Laura E.; Kasperkiewicz, Paulina; Drag, Marcin; Kälvälä, Sara; Moisio, Eemeli; Savinainen, Juha R.; Laitinen, Jarmo T. (2020)
    Background Serine hydrolases (SHs) are a functionally diverse family of enzymes playing pivotal roles in health and disease and have emerged as important therapeutic targets in many clinical conditions. Activity-based protein profiling (ABPP) using fluorophosphonate (FP) probes has been a powerful chemoproteomic approach in studies unveiling roles of SHs in various biological systems. ABPP utilizes cell/tissue proteomes and features the FP-warhead, linked to a fluorescent reporter for in-gel fluorescence imaging or a biotin tag for streptavidin enrichment and LC-MS/MS-based target identification. Existing ABPP approaches characterize global SH activity based on mobility in gel or MS-based target identification and cannot reveal the identity of the cell-type responsible for an individual SH activity originating from complex proteomes. Results Here, by using an activity probe with broad reactivity towards the SH family, we advance the ABPP methodology to glioma brain cryosections, enabling for the first time high-resolution confocal fluorescence imaging of global SH activity in the tumor microenvironment. Tumor-associated cell types were identified by extensive immunohistochemistry on activity probe-labeled sections. Tissue-ABPP indicated heightened SH activity in glioma vs. normal brain and unveiled activity hotspots originating from tumor-associated neutrophils (TANs), rather than tumor-associated macrophages (TAMs). Thorough optimization and validation was provided by parallel gel-based ABPP combined with LC-MS/MS-based target verification. Conclusions Our study advances the ABPP methodology to tissue sections, enabling high-resolution confocal fluorescence imaging of global SH activity in anatomically preserved complex native cellular environment. To achieve global portrait of SH activity throughout the section, a probe with broad reactivity towards the SH family members was employed. As ABPP requires no a priori knowledge of the identity of the target, we envisage no imaginable reason why the presently described approach would not work for sections regardless of species and tissue source.
  • Rantamäki, Antti; Chen, Wen; Hyväri, Paulus; Helminen, Jussi; Partl, Gabriel; King, Alistair W. T.; Wiedmer, Susanne (2019)
    Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder - increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures.
  • Karhu, Kristiina; Hilasvuori, Emmi; Jarvenpaa, Marko; Arppe, Laura; Christensen, Bent T.; Fritze, Hannu; Kulmala, Liisa; Oinonen, Markku; Pitkanen, Juha-Matti; Vanhala, Pekka; Heinonsalo, Jussi; Liski, Jari (2019)
    Most of the carbon (C) stored in temperate arable soils is present in organic matter (OM) intimately associated with soil minerals and with slow turnover rates. The sensitivity of mineral-associated OM to changes in temperature is crucial for reliable predictions of the response of soil C turnover to global warming and the associated flux of carbon dioxide (CO2) from the soil to the atmosphere. We studied the temperature sensitivity of C in <63 mu m fractions rich in mineral-associated organic matter (MOM) and of C in > 63 mu m fractions rich in particulate organic matter (POM). The fractions were isolated by physical separation of two light-textured arable soils where the C4-plant silage maize had replaced C3-crops 25 years ago. Differences in C-13 abundance allowed for calculation of the age of C in the soil-size fractions (old C, C3-C > 25 years; recent C, C4-C <25 years). We incubated bulk soils ( <2 mm) and size fractions sequentially at 6, 18, 26 and 34 degrees C (ramping up and down the temperature scale) and calculated the temperature sensitivity of old and recent C from (CO2)-C-12 and (CO2)-C-13 evolution rates. The temperature sensitivity was similar or slightly higher for POM than for MOM. Within the POM fraction, old C3-C was more sensitive to changes in temperature than recent C4-C. For the MOM fraction, the temperature sensitivity was unrelated to the age of C. Quantitative PCR analysis indicated that the proportions of bacteria, archaea and fungi did not change during incubation. Our results suggest that while OM stabilizing mechanisms affect the temperature sensitivity of soil C, temperature sensitivity appears unrelated to the age of mineral-associated OM.