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  • 1.
    B. Kumar, Ramakrishnan
    et al.
    Karolinska institutet, Sverige.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska institutet, Sverige.
    Jegerschöld, Caroline
    Karolinska institutet, Sverige.
    Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy2017In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 121, article id e55148Article in journal (Refereed)
    Abstract [en]

    Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

  • 2. Bello, M. A.
    et al.
    Ruiz-León, Y.
    Sandoval-Sierra, J. V.
    Rezinciuc, Svetlana
    KTH, School of Biotechnology (BIO), Glycoscience.
    Diéguez-Uribeondo, J.
    Scanning electron microscopy (SEM) protocols for problematic plant, oomycete, and fungal samples2017In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, Vol. 2017, no 120, article id e55031Article in journal (Refereed)
    Abstract [en]

    Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricalesas examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

  • 3.
    Dias, Jorge. T.
    et al.
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Svedberg, Gustav
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Nystrand, M.
    Svahn Andersson, Helene
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Gantelius, Jesper
    KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Rapid nanoprobe signal enhancement by in situ gold nanoparticle synthesis2018In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, Vol. 2018, no 133, article id e57297Article in journal (Refereed)
    Abstract [en]

    The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good's buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera. 

  • 4. Gallstedt, Mikael
    et al.
    Pettersson, Henrik
    Johansson, Therese
    Newson, William R.
    Johansson, Eva
    Hedenqvist, Mikael S.
    KTH, School of Chemical Science and Engineering (CHE), Fibre and Polymer Technology.
    Film Extrusion of Crambe abyssinica/Wheat Gluten Blends2017In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 119, article id e54770Article in journal (Refereed)
    Abstract [en]

    Crambe abyssinica is a plant with potential for use in industrial (non-food) plant oil production. The side stream from this oil production is a high-protein crambe meal that has limited value, as it is not fit for food or feed use. However, it contains proteins that could potentially make it a suitable raw material for higher-value products. The purpose of this study was to find methods of making this side stream into extruded films, showing that products with a higher value can be produced. The study mainly considered the development of material compositions and methods of preparing and extruding the material. Wheat gluten was added as a supportive protein matrix material, together with glycerol as a plasticizer and urea as a denaturant. The extrudate was evaluated with respect to mechanical (tensile testing) and oxygen barrier properties, and the extrudate structure was revealed visually and by scanning electron microscopy. A denser, more homogeneous material had a lower oxygen transmission rate, higher strength, and higher extensibility. The most homogeneous films were made at an extruder die temperature of 125-130 degrees C. It is shown here that a film can be extruded with promising mechanical and oxygen barrier properties, the latter especially after a final compression molding step.

  • 5. Katchy, Anne
    et al.
    Williams, Cecilia
    Profiling of estrogen-regulated microRNAs in breast cancer cells.2014In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 84, article id e51285Article in journal (Refereed)
    Abstract [en]

    Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ERα, and ERβ. ERα is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ERα's regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ERα-positive breast cancer cell lines were compared before and after estrogen-activation using both the µParaflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations, and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

  • 6.
    Kürten, Charlotte
    et al.
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Syren, Per-Olof
    KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Biotechnology (BIO), Proteomics and Nanobiotechnology.
    Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes2016In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 107, article id e53168Article in journal (Refereed)
    Abstract [en]

    Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

  • 7. Ruprecht, Colin
    et al.
    Tohge, Takayuki
    Fernie, Alisdair
    Mortimer, Cara L.
    Kozlo, Amanda
    Fraser, Paul D.
    Funke, Norma
    Cesarino, Igor
    Vanholme, Ruben
    Boerjan, Wout
    Morreel, Kris
    Burgert, Ingo
    Gierlinger, Notburga
    Bulone, Vincent
    KTH, School of Biotechnology (BIO), Glycoscience.
    Schneider, Vera
    Stockero, Andrea
    Navarro, Juan Pedro
    Pudel, Frank
    Tambuyser, Bart
    Hygate, James
    Bumstead, Jon
    Notley, Louis
    Persson, Staffan
    Transcript and Metabolite Profiling for the Evaluation of Tobacco Tree and Poplar as Feedstock for the Bio-based Industry2014In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 87, p. e51393-Article in journal (Refereed)
    Abstract [en]

    The global demand for food, feed, energy and water poses extraordinary challenges for future generations. It is evident that robust platforms for the exploration of renewable resources are necessary to overcome these challenges. Within the multinational framework MultiBioPro we are developing biorefinery pipelines to maximize the use of plant biomass. More specifically, we use poplar and tobacco tree (Nicotiana glauca) as target crop species for improving saccharification, isoprenoid, long chain hydrocarbon contents, fiber quality, and suberin and lignin contents. The methods used to obtain these outputs include GC-MS, LC-MS and RNA sequencing platforms. The metabolite pipelines are well established tools to generate these types of data, but also have the limitations in that only well characterized metabolites can be used. The deep sequencing will allow us to include all transcripts present during the developmental stages of the tobacco tree leaf, but has to be mapped back to the sequence of Nicotiana tabacum. With these set-ups, we aim at a basic understanding for underlying processes and at establishing an industrial framework to exploit the outcomes. In a more long term perspective, we believe that data generated here will provide means for a sustainable biorefinery process using poplar and tobacco tree as raw material. To date the basal level of metabolites in the samples have been analyzed and the protocols utilized are provided in this article.

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  • nn-NO
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