103 research outputs found
Structural Investigations on Lithium-Doped Protic and Aprotic Ionic Liquids
Solutions
of lithium bisÂ(trifluoromethanesulfonyl)Âimide (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>
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>
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
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
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
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
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
MOESM1 of Focal colorectal uptake in 18FDG-PET/CT: maximum standard uptake value as a trigger in a semi-automated screening setting
Additional file 1: Table S1. Demographic Data of Patients with Colorectal Cancer (n=54)
IL-6 Amplifies TLR Mediated Cytokine and Chemokine Production: Implications for the Pathogenesis of Rheumatic Inflammatory Diseases
<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.
<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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