103 research outputs found

    Structural Investigations on Lithium-Doped Protic and Aprotic Ionic Liquids

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    Solutions of lithium bis­(trifluoromethanesulfonyl)­imide (LiNTf<sub>2</sub>), in four different [NTf<sub>2</sub>]<sup>−</sup>-based ionic liquids, are extensively investigated as potential electrolytes for lithium-ion batteries. Solvation of the [Li]<sup>+</sup> ions in the ionic liquids and its impact on their physicochemical properties are studied herein with the aid of molecular dynamics simulations. The cationic components of the investigated liquids were systematically varied so as to individually evaluate effects of specific structural changes; increase in ring size, the addition of an alkyl chain and absence of an acidic proton, on the solvation and mobility of the [Li]<sup>+</sup> cations. The studied cations also allow for a direct comparison between solutions of [Li]<sup>+</sup> salt in protic and aprotic ionic liquids. Emphasis is laid on elucidating the interactions between the [Li]<sup>+</sup> and [NTf<sub>2</sub>]<sup>−</sup> ions revealing slightly higher coordination numbers for the aprotic solvent, benchmarked against experimental measurements. The study suggests that the ionic liquids largely retain their structure upon salt addition, with interactions within the liquids only slightly perturbed. The rattling motion of the [Li]<sup>+</sup> cations within cages formed by the surrounding [NTf<sub>2</sub>]<sup>−</sup> anions is examined by the analysis of [Li]<sup>+</sup> autocorrelation functions. Overall, the solvation mechanism of [Li]<sup>+</sup> salt, within the hydrogen-bonded network of the ionic liquids, is detailed from classical and <i>ab initio</i> molecular dynamics simulations

    Synthetic Core Promoters for <i>Pichia pastoris</i>

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    Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural <i>AOX1</i> promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields

    Synthetic Core Promoters for <i>Pichia pastoris</i>

    No full text
    Synthetic promoters are commonly used tools for circuit design or high level protein production. Promoter engineering efforts in yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory sequences such as transcription factor binding sites. In higher eukaryotes synthetic core promoters, directly needed for transcription initiation by RNA Polymerase II, have been successfully designed. Here we report the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this synthetic core promoter sequence to engineer the core promoter of the natural <i>AOX1</i> promoter, thereby creating a set of core promoters providing a range of different expression levels. As opposed to engineering strategies of the significantly longer entire promoter, such short core promoters can directly be added on a PCR primer facilitating library generation and are sufficient to obtain variable expression yields

    Prozesstechnik der DME und OME-Synthese und Life Cycle Assessment

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    Table S1. Primers used in this study and detailed plasmid construction. Primers used for creating the S. pombe and P. pastoris plasmids are separated. Also primers for construction of the plasmids are separated from primers for sequencing and insertion of GOIs. In addition separate spreadsheets are providing information on the exact construction of the plasmids by listing the PCR products and restriction enzymes used for assembly

    Additional file 2: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

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    Figure S2. Simple strategy for confirming the orientation of the insert. The forward or reverse primer used for amplifying the insert can be used together with the forward or reverse sequencing primer of the vector to confirm the correct orientation. Upon correct primer choice only the forward orientation gives a PCR fragment. The sequencing primers designed for Sanger sequencing allow sequencing of the insert from both sides. Depending on the vector, different primers should be used (e.g. when the MFalpha signal sequence or a fusion protein is present, see the primer list for all sequencing primers available)

    Additional file 4: of Restriction site free cloning (RSFC) plasmid family for seamless, sequence independent cloning in Pichia pastoris

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    Figure S3. Fluorescence measurements of fusions of HRP to eGFP. Samples are labeled in the same way as in Figure 4. eGFP fluorescence of supernatants and cell pellets of methanol induced cells were normalized per cell density (OD600)

    Synthetic Core Promoters as Universal Parts for Fine-Tuning Expression in Different Yeast Species

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    Synthetic biology and metabolic engineering experiments frequently require the fine-tuning of gene expression to balance and optimize protein levels of regulators or metabolic enzymes. A key concept of synthetic biology is the development of modular parts that can be used in different contexts. Here, we have applied a computational multifactor design approach to generate <i>de novo</i> synthetic core promoters and 5′ untranslated regions (UTRs) for yeast cells. In contrast to upstream <i>cis</i>-regulatory modules (CRMs), core promoters are typically not subject to specific regulation, making them ideal engineering targets for gene expression fine-tuning. 112 synthetic core promoter sequences were designed on the basis of the sequence/function relationship of natural core promoters, nucleosome occupancy and the presence of short motifs. The synthetic core promoters were fused to the <i>Pichia pastoris AOX1</i> CRM, and the resulting activity spanned more than a 200-fold range (0.3% to 70.6% of the wild type <i>AOX1</i> level). The top-ten synthetic core promoters with highest activity were fused to six additional CRMs (three in <i>P. pastoris</i> and three in <i>Saccharomyces cerevisiae</i>). Inducible CRM constructs showed significantly higher activity than constitutive CRMs, reaching up to 176% of natural core promoters. Comparing the activity of the same synthetic core promoters fused to different CRMs revealed high correlations only for CRMs within the same organism. These data suggest that modularity is maintained to some extent but only within the same organism. Due to the conserved role of eukaryotic core promoters, this rational design concept may be transferred to other organisms as a generic engineering tool

    IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases

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    <div><p>The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.</p></div

    Exposure to IL-6 enhances cytokine and chemokine production in SFMC.

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    <p>SFMC were left to adhere on plastic for 3 hours in DMEM supplemented with 10% fetal calf serum (FCS). SFMC were pre-exposed to IL-6/sIL-6R in combination with IgG1 or with TCZ for 1 hour. Cells were then stimulated for 18 hours with LPS (10 ng/ml) (<b>A</b>), and S100A8 (5 µg/ml) (<b>B</b>). IL-1β, CXCL8 and CCL2 levels were measured by ELISA. *p<0.05 for values from IL-6/sIL-6R -stimulated compared with NT cells.</p
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