Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRN

Monitoring mis‐acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chemically aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site‐specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run‐off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amounts of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately determine suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser‐scanning confocal microscopy. Control experiments showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl‐tRNA synthetases. This reporter assay opens the way for the optimization of essential experimental parameters for expanding the scope of the suppressor tRNA technology to different cell type

Monitoring mis-acylated tRNA suppression efficiency in mammalian cells via EGFP fluorescence recovery

A reporter assay was developed to detect and quantify nonsense codon suppression by chem. aminoacylated tRNAs in mammalian cells. It is based on the cellular expression of the enhanced green fluorescent protein (EGFP) as a reporter for the site-specific amino acid incorporation in its sequence using an orthogonal suppressor tRNA derived from Escherichia coli. Suppression of an engineered amber codon at position 64 in the EGFP run-off transcript could be achieved by the incorporation of a leucine via an in vitro aminoacylated suppressor tRNA. Microinjection of defined amts. of mutagenized EGFP mRNA and suppressor tRNA into individual cells allowed us to accurately det. suppression efficiencies by measuring the EGFP fluorescence intensity in individual cells using laser-scanning confocal microscopy. Control expts. showed the absence of natural suppression or aminoacylation of the synthetic tRNA by endogenous aminoacyl-tRNA synthetases. This reporter assay opens the way for the optimization of essential exptl. parameters for expanding the scope of the suppressor tRNA technol. to different cell types. [on SciFinder (R)

Print‐Light‐Synthesis of Gold Thin Film Electrodes for Electrochemical Sensing

The one-step fabrication of gold films by inkjet printing of a gold precursor ink and its photochemical reduction by exposure to UV light is presented. Inkjet printing creates on a substrate with high control micrometer-thin reaction volumes in which upon direct high-intensity light irradiation, the gold precursor reduces to pure and well-adhered Au particles, while all other ink components escape in the gas phase, without the need for any further post-treatment. The Au precursor ink does neither contain stabilizing agents, such as polymers or surfactants, nor sacrificial compounds, such as photoinitiators, to initiate and accelerate the reduction. This economic and green process is known as Print-Light-Synthesis (PLS) and is herein used to create gold patterns of thin compact Au films and separated Au nanoparticles. Gold loadings are in the μg cm−2 range and precisely controlled, thanks to the inkjet printing parameters. The gold films are characterized by spectroscopic and electrochemical methods. Finally, the applicability of Au films as electrochemical sensors is demonstrated for the detection of 1,4-butanediol in comparison to a commercial screen-printed Au electrode. The PLS Au electrode shows a 20 times higher sensitivity and opens new possibilities for disposable electrode production

Insertion of T4-lysozyme (T4L) can be a useful tool for studying olfactory-related GPCRs.

The detergents used to solubilize GPCRs can make crystal growth the rate-limiting step in determining their structure. The Kobilka laboratory showed that insertion of T4-lysozyme (T4L) in the 3rd intracellular loop is a promising strategy towards increasing the solvent-exposed receptor area, and hence the number of possible lattice-forming contacts. The potential to use T4L with the olfactory-related receptors hOR17-4 and hVN1R1 was thus tested. The structure and function of native and T4L-variants were compared. Both receptors localized to the cell membrane, and could initiate ligand-activated signaling. Purified receptors not only had the predicted alpha-helical structures, but also bound their ligands canthoxal (MW = 178.23) and myrtenal (MW = 150.22). Interestingly, the T4L variants had higher percentages of soluble monomers compared to protein aggregates, effectively increasing the protein yield that could be used for structural and function studies. They also bound their ligands for longer times, suggesting higher receptor stability. Our results indicate that a T4L insertion may be a general method for obtaining GPCRs suitable for structural studies

Monitoring proliferative activities of hormone-like odorants in human breast cancer cells by gene transcription profiling and electrical impedance spectroscopy

The human estrogen receptor alpha (ER!) mediates the proliferative action of hormones in breast cancer cells by regulating the expression of target genes to control cellular functions. Current methodologies do not permit a real-time assessment of these processes in living cells. We overcome this limitation using electrical cell-substrate impedance sensing for measuring ER!-regulated signaling processes indicative of the onset of cell proliferation to target them for compound screenings. We report that hormone like odorants regulate, similarly as natural estrogen, ER!-mediated gene expression involved in mitogenic and developmental processes in MCF7 breast cancer cells. An odorant concentration-dependent switch in cell responses was detectable already 10–15 h post-stimulation, providing rapid quanti!cation of hormonal activity before cell division occurred. Though ER! exhibits complex regulatory roles our noninvasive approach captures its activity for accelerated screenings of compounds promoting breast cancer cell proliferation expanding the analysis of ER! signaling networks

Aluminium Foil as Single-Use Substrate for MALDI-MS Fingerprinting of Different Melanoma Cell Lines

Herein, we present the intact cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the fingerprinting of human melanoma cancer cell lines grown on aluminium foil. To perform the MALDI-MS assay, melanoma cells were cultured on a flat and thin foil, which was directly transferred to the target plate of MALDI-MS for analysis. The influence of a wide range of cell fixation protocols (i.e. formalin-based and alcohol-based methods) and MALDI matrices on the obtained characteristic spectra was investigated. For the optimization of the MALDI-MS protocol, the MS fingerprints of the melanoma WM-239 cell line with and without an overexpressed enhanced green fluorescent protein were employed. The fingerprints obtained from WM-239 cells grown on aluminum foil were compared with intact cell MALDI of cell pellet and presented higher sensitivity in high m/z range. The optimized protocol was subsequently applied to characterise melanoma cell lines derived from different cancer stages and allowed the identification of unique MS signals that can be used for the differentiation between the studied cell lines (i.e. molecular weight equal to 10.0 kDa and 26.1 kDa)

Electrostatic Spray Ionization Mass Spectrometry Imaging

Imaging samples on a surface by mass spectrometry (MS) requires the combination of MS detection with a scanning mode that enables localized desorption and ionization and/or detection of sample analytes with good spatial resolution. We have developed a new mass spectrometry imaging (MSI) method based on electrostatic spray ionization. It works under ambient conditions and can be applied to a wide range of molecules providing quantitative MS analysis even in the presence of salts in excess. 2D MS images of protein and peptide spots, inkjet-printed black dye patterns, and cells were obtained. The presented novel ambient ionization mass spectrometry imaging method can find many applications in analytical and bioanalytical chemistr

Effective removal of the rotifer Brachionus calyciflorus from a Chlorella vulgaris microalgal culture by homogeneous solar photo-Fenton at neutral pH

In this study, a citrate-modified photo-Fenton process was successfully applied to decontaminate a Chlorella vulgaris microalgae culture spiked with the rotifer Brachionus calyciflorus (5 individuals mL−1). The applied treatment (1 mg L−1 Fe2+, 20 mg L−1 H2O2, 17.5 mg L−1 citric acid) had only moderate effects on viability and regrowth of the microalgae since, after a short post-treatment delay of a few days, they reached final cell densities similar to that obtained for microalgae cultures that were not spiked. The decontamination was effective as no regrowth of rotifers was observed in the microalgae cultures after treatment. The efficacy of the citrate-modified photo-Fenton treatment was also studied with a higher starting concentration of 20 rotifers mL−1 and was compared with a solar light/H2O2 treatment. Results show that both treatments had similar efficacies on the rotifer elimination, but that the citrate-modified photo-Fenton treatment had a lower negative impact on the regrowth of microalgae than the solar light/H2O2 treatment. However, when microalgae cultures were spiked with 20 rotifers mL−1, rotifers were only partially inactivated and post-treatment regrowth occurred, which highlights the importance to apply the photo-Fenton process at an early stage of a contamination to achieve full rotifer elimination. In any case, a contamination with 5 rotifers mL−1 is already a significant threat as numbers above 1000 rotifers mL−1 were reached after 14 days and caused the microalgae culture to fail. Overall, our treatment suggests that the citrate-modified solar photo-Fenton process is an environmentally friendly solution to support the maintenance of contaminant-free microalgal cultures

Untersuchung der Tyrosinase-Expression in nicht-metastatischen und metastatischen Melanomgeweben durch elektrochemische Rastersondenmikroskopie

Trotz des enormen Fortschritts, der bei der Diagnose von Melanomen erzielt wurde, bleibt die zuverlässige Erkennung der verschiedenen Malignomstadien eine Herausforderung. Derzeitige Strategien setzen auf die optische Erfassung der Konzentration und räumlichen Verteilung spezifischer Biomarker. Neueste optische Methoden können allerdings durch Interferenzen mit Hintergrundfarben und durch Autofluoreszenz beeinflusst werden. Wir bewältigen diese Defizite hier unter Einsatz der elektrochemischen Rastersondenmikroskopie (SECM), um den prognostischen Indikator Tyrosinase (TyR) in nicht-metastatischen und metastatischen Melanomgeweben mithilfe einer weichen Mikroelektrodensonde abzubilden. Das elektrochemische Auslesen der TyR-Verteilung wurde durch das Adaptieren eines immunchemischen Verfahrens ermöglicht. Wir zeigen, dass die SECM die erwähnten Beschränkungen optischer Methoden übertreffen kann und bisher nicht gekannte Möglichkeiten für eine verbesserte Diagnose und das Verständnis der räumlichen Verteilung von TyR in verschiedenen Melanomstadien eröffnet