Exploring in-plane interactions beside an adsorbed molecule with lateral force microscopy.

Atomic force microscopy with a CO-functionalized tip can be used to directly image the internal structure of a planar molecule and to characterize chemical bonds. However, hydrogen atoms usually cannot be directly observed due to their small size. At the same time, these atoms are highly important, since they can direct on-surface chemical reactions. Measuring in-plane interactions at the sides of PTCDA (3,4,9,10-perylenetetracarboxylic dianhydride) molecules with lateral force microscopy allowed us to directly identify hydrogen atoms via their repulsive signature, which we confirmed with a model incorporating radially symmetric atomic interactions. Additional features were observed in the force data and could not be explained by H-bonding of the CO tip with the PTCDA sides. Instead, they are caused by electrostatic interaction of the large dipole of the metal apex, which we verified with density functional theory. This calculation allowed us to estimate the strength of the dipole at the metal tip apex. To further confirm that this dipole generally affects measurements on weakly polarized systems, we investigated the archetypical surface adsorbate of a single CO molecule. We determined the radially symmetric atomic interaction to be valid over a large solid angle of 5.4 sr, corresponding to 82°. We therefore find that in both the PTCDA and CO systems, the underlying interaction preventing direct observations of H-bonding and causing a collapse of the radially symmetric model is the dipole at the metal apex, which plays a significant role when approaching closer than standard imaging heights.

Senescence in a cell culture model for burn wounds.

Thermal injuries cause severe damage on the cellular and tissue level and are considered especially challenging in the clinical routine. Complex interactions of different cell types and pathways dictate the formation of burn wounds. Thus, complications like burn wound progression, where so far viable tissue becomes necrotic and the size and depth of the wound increases, are difficult to explain, mainly due to the lack of simple model systems. We tested the behavior of human fibroblasts after heat treatment. A prominent response of the cells is to activate the heat shock response (HSR), which is one of the primary emergency mechanisms of the cell to proteotoxic stress factors such as heat. However, after a powerful but not lethal heat shock we observed a delayed activation of the HSR. Extending this model system, we further investigated these static cells and observed the emergence of senescent cells. In particular, the cells became ß-galactosidase positive, increased p16 levels and developed a senescence-associated secretory phenotype (SASP). The secretion of cytokines like IL-6 is reminiscent of burn wounds and generates a bystander effect in so far non-senescent cells. In agreement with burn wounds, a wave of cytokine secretion enhanced by invading immune cells could explain complications like burn wound progression. A simple cell culture model can thus be applied for the analysis of highly complex conditions in human tissues.

Strumming a Single Chemical Bond.

Atomic force microscopy and scanning tunneling microscopy can image the internal structure of molecules adsorbed on surfaces. One reliable method is to terminate the tip with a nonreactive adsorbate, often a single CO molecule, and to collect data at a close distance where Pauli repulsion plays a strong role. Lateral force microscopy, in which the tip oscillates laterally, probes similar interactions but has the unique ability to pull the CO over a chemical bond, load it as a torsional spring, and release it as it snaps over with each oscillation cycle. This produces measurable energy dissipation. The dissipation has a characteristic decay length in the vertical direction of 4 pm, which is 13 times smaller than the decay length in typical STM or AFM experiments.

A dual luciferase assay for evaluation of skin sensitizing potential of medical devices.

According to standing regulations animal tests are still state of the art for the evaluation of the sensitization potential of medical devices. The aim of our study was to develop an in vitro method that can be used for testing of extracts of medical devices. The novel MDA-ARE assay is a cell based reporter gene assay focused on the ARE-Nrf2 pathway, which is involved in the dermal sensitization process. Optimization of the reporter construct and the cell line resulted in an improvement of the detection limit and a reduction of the incubation time to 6 h, which lowers cytotoxic side effects of the extracts on the cells. Using the assay, 21 out of 22 pure chemicals were identified correctly as skin sensitizers or non-sensitizers. All sensitizers could be detected at far lower concentrations compared to the local lymph node assay, the state-of-the-art animal test. To evaluate the assay's suitability for the testing of medical devices, medical grade silicone containing 0.1% of known skin sensitizers was prepared as positive controls and extracts of these positive controls were tested in comparison to extracts from pure silicone samples. All silicone samples were correctly and reproducibly identified as sensitizing or non-sensitizing demonstrating that the MDA-ARE assay is a sensitive and reliable tool for the detection of skin sensitizers in extracts of medical devices. The developed and validated test protocol was used for medical device extracts and showed its applicability for real samples and thus can contribute to reduce or even to replace the need for animal tests.

The function of tcf3 in medaka embryos: efficient knockdown with pePNAs.

BACKGROUND: The application of antisense molecules, such as morpholino oligonucleotides, is an efficient method of gene inactivation in vivo. We recently introduced phosphonic ester modified peptide nucleic acids (PNA) for in vivo loss-of-function experiments in medaka embryos. Here we tested novel modifications of the PNA backbone to knockdown the medaka tcf3 gene. RESULTS: A single tcf3 gene exists in the medaka genome and its inactivation strongly affected eye development of the embryos, leading to size reduction and anophthalmia in severe cases. The function of Tcf3 strongly depends on co-repressor interactions. We found interactions with Groucho/Tle proteins to be most important for eye development. Using a dominant negative approach for combined inactivation of all groucho/tle genes also resulted in eye phenotypes, as did interference with three individual tle genes. CONCLUSIONS: Our results show that side chain modified PNAs come close to the knockdown efficiency of morpholino oligonucleotides in vivo. A single medaka tcf3 gene combines the function of the two zebrafish paralogs hdl and tcf3b. In combination with Groucho/Tle corepressor proteins Tcf3 acts in anterior development and is critical for eye formation.

Repression motif in HSF1 regulated by phosphorylation.

The heat shock factor 1 (HSF1) is a transcription factor that itself is a sensor for stress and integrates various intrinsic or environmental stress sensing pathways. Thus HSF1 orchestrates the heat shock response (HSR) by translating these pathways into a distinct transcriptional program that aids the cells to cope with and adapt to proteotoxic stress. Although heavily researched the regulation of HSF1 activation is still not completely understood. A conserved reaction to stress is the hyperphosphorylation of the otherwise confined constitutive phosphorylated HSF1. Therefore, this stress specific phosphorylation is believed to be involved in the regulatory mechanism and hence, was and is focus of many studies, ascribing various effects to single phosphorylation sites. To gain additional insight into effects of phosphorylation, HSF1 carrying amino acid substitutions on up to 18 amino acids were tested for their transactivation potential on an HSR reporter plasmid. A pattern of eleven phosphor-mimicking and diminishing amino acid substitutions on well-known phosphorylation sites of HSF1 were introduced to produce transcriptional active [11 M(+)] or repressed [11 M(-)] phenotypes. It could be confirmed that heat activates HSF1 regardless of phosphorylation. Distinct cellular stress, obtained by chemical HSR inducers or mimicked by a constitutively active HSF1, showed clear differences in the activation potential of HSF1-11 M(+) and 11 M(-). Further refinement to the single amino acid level identified the S303/307 double-phosphorylation motif, wherein phosphorylation of S303 was sole responsible for the repressing effect. The effect could be reproduced in different cell lines and is not entirely based on degradation. A small repression motif could be dissociated from the HSF1 context, which is still capable of repressing the background transcription of a specifically designed reporter plasmid. Taken together these results indicate, that besides already described mechanisms of pS303/307 mediated repression of HSF1 activation, an additional mechanism repressing the transcriptional output of the entire HSE containing promoter is mediated by this small repressive motif.

Quantitative Comparison of HSF1 Activators.

The heat shock response (HSR) pathway is a highly conserved rescue mechanism, which protects the cells from harmful insults disturbing the cellular protein homeostasis via expression of chaperones. Furthermore, it was demonstrated to play crucial roles in various diseases like neurodegeneration and cancer. For neurodegenerative diseases, an overexpression of chaperones is a potential therapeutic approach to clear the cells from non-functional protein aggregates. Therefore, activators of the HSR pathway and its master regulator HSF1 are under close observation. There are numerous HSR activators published in the literature using different model systems, experimental designs, and readout assays. The aim of this work was to provide a quantitative comparison of a broad range of published activators using a newly developed HSF responsive dual-luciferase cell line. Contrary to natural target genes, which are regulated by multiple input pathways, the artificial reporter exclusively reacts to HSF activity. In addition, the results were compared to endogenous heat shock protein expression. As a result, great differences in the intensity of pathway activation were observed. In addition, a parallel viability assessment revealed high variability in the specificity of the drugs. Furthermore, the differences seen compared to published data indicate that some activators exhibit tissue-specific differences leading to interesting assumptions about the regulation of HSF1.

Lateral Force Microscopy Reveals the Energy Barrier of a Molecular Switch.

Copper phthalocyanine (CuPc) is a small molecule often used in organic light emitting diodes where it is deposited on a conducting electrode. Previous scanning tunneling microscopy (STM) studies of CuPc on Cu(111) have shown that inelastic tunneling events can cause CuPc to switch between a ground state and two symmetrically equivalent metastable states in which the molecule is rotated. We investigated CuPc on Cu(111) and Ag(111) with STM and lateral force microscopy (LFM). Even without inelastic events, the presence of the tip can induce rotations and upon closer approach, causes the rotated states to be favored. Combining STM measurements at various temperatures and LFM measurements, we show that the long-range attraction of the tip changes the potential energy landscape of this molecular switch. We can also determine the geometry of the rotated and ground states. We compare our observations of CuPc on Cu(111) to CuPc on Ag(111). On Ag(111), CuPc appears flat and does not rotate. Stronger bonding typically involves shorter bond lengths, larger shifts of energy levels, and structural stability. Although the binding of CuPc to Cu(111) is stronger than that on Ag(111), the nonplanar geometry of CuPc on Cu(111) is accompanied by two metastable states which are not present on the Ag(111) surface.

HepGentox: a novel promising HepG2 reportergene-assay for the detection of genotoxic substances in complex mixtures.

BACKGROUND: In risk assessment, genotoxicity is a key factor to determine the safety for the consumer. Most in vitro genotoxicity assays were developed for the assessment of pure substances. However, in recent years more attention has been given to complex mixtures, where usually low amounts of a substance are present. For high-throughput screening, a toxicologically sensitive assay should be used, covering a broad range of genotoxic substances and detecting them at low concentrations. HepG2 cells have been recommended as one of the prime candidates for genotoxicity testing, as they are p53 competent, less prone towards cytotoxic effects and tend to have some metabolic activity. METHODS: A HepG2 liver cell line was characterized for its suitability for genotoxicity assessment. For this, a luciferase based reporter gene assay revolving around the p53 pathway was validated for the analysis of pure substances and of complex mixtures. Further, the cell's capability to detect genotoxins correctly with and without an exogenous metabolizing system, namely rat liver S9, was assessed. RESULTS: The assay proved to have a high toxicological sensitivity (87.5%) and specificity (94%). Further, the endogenous metabolizing system of the HepG2 cells was able to detect some genotoxins, which are known to depend on an enzymatic system. When complex mixtures were added this did not lead to any adverse effects concerning the assays performance and cytotoxicity was not an issue. DISCUSSION: The HepGentox proved to have a high toxicological sensitivity and specificity for the tested substances, with similar or even lower lowest effective concentration (LEC) values, compared to other regulatory mammalian assays. This combines some important aspects in one test system, while also being less time and material consuming and covering several genotoxicity endpoints. As the assay performs well with and without an exogenous metabolizing system, no animal liver fractions have to be used, which application is discussed controversially and is considered to be expensive and laborious in sample testing. Because of this, the HepGentox is suitable for a cost-efficient first screening approach to obtain important information with human cells for further approaches, with a relatively fast and easy method. Therefore, the HepGentox is a promising assay to detect genotoxic substances correctly in complex mixtures even at low concentrations, with the potential for a high throughput application. In a nutshell, as part of an in vitro bioassay test battery, this assay could provide valuable information for complex mixtures.

Evaluation of the Suitability of Mammalian In Vitro Assays to Assess the Genotoxic Potential of Food Contact Materials.

BACKGROUND: Non-targeted screening of food contact materials (FCM) for non-intentionally added substances (NIAS) reveals a great number of unknown and unidentified substances present at low concentrations. In the absence of toxicological data, the application of the threshold of toxicological concern (TTC) or of EU Regulation 10/2011 requires methods able to fulfill safety threshold criteria. In this review, mammalian in vitro genotoxicity assays are analyzed for their ability to detect DNA-damaging substances at limits of biological detection (LOBD) corresponding to the appropriate safety thresholds. RESULTS: The ability of the assays to detect genotoxic effects varies greatly between substance classes. Especially for direct-acting mutagens, the assays lacked the ability to detect most DNA reactive substances below the threshold of 10 ppb, making them unsuitable to pick up potential genotoxicants present in FCM migrates. However, suitability for the detection of chromosomal damage or investigation of other modes of action makes them a complementary tool as part of a standard test battery aimed at giving additional information to ensure safety. CONCLUSION: improvements are necessary to comply with regulatory thresholds to consider mammalian genotoxicity in vitro assays to assess FCM safety.

Suitability of the Ames test to characterise genotoxicity of food contact material migrates.

Non-intentionally added substances (NIAS) are chemical impurities which can migrate from packaging materials (FCM) into food. Safety assessment of NIAS is required by European law, but currently there is no comprehensive testing strategy available. In this context, one key element is to get insight on the potential presence of genotoxic NIAS in FCM migrates. This raises questions about the limit at which genotoxins can be detected in complex mixtures such as FCM migrates, and if such limits of detection (LOD) would be compatible with safety. In this context, the present review assesses the suitability of the Ames assay to address genotoxicity of FCM migrates. Lowest effective concentrations of packaging-related and other chemicals in test media were retrieved from scientific literature and used as surrogates of LODs to be benchmarked against a value of 0.01 mg kg-1 (10 ppb) in migrates. This is a pragmatic threshold used in FCM safety evaluation to prioritise substances requiring proper identification and risk assessment. The analysis of the data shows that only potent genotoxins can theoretically be detectable at a level of 0.01 mg kg-1 in migrates or food. Only a minority (10%) of genotoxic chemicals reported to be associated with FCMs could be picked up at a level of 0.01 mg kg-1 or lower. Overall, this review shows that the Ames test in its present form cannot be used as standalone method for evaluating the genotoxic potential of FCM migrates, but must be used together with other information from analytical chemistry and FCM manufacturing.

HSF1 mediated stress response of heavy metals.

The heat shock response (HSR) pathway is a highly conserved cellular stress response and mediated by its master regulator HSF1. Activation of the pathway results in the expression of chaperone proteins (heat shock proteins; HSP) to maintain protein homeostasis. One of the genes strongest upregulated upon stress is HSPA1A (HSP72). Heavy metals are highly toxic to living organisms and known as environmental contaminants, due to industrialisation. Furthermore, many of them are well-described inducers of the HSR pathway. Here we compare the effect of different heavy metals, concerning their potential to activate HSF1 with a sensitive artificial heat shock reporter cell line, consisting of heat shock elements (HSE). In general the responses of the artificial promoter to heavy metal stress were in good agreement with those of well-established HSF1 target genes, like HSPA1A. Nevertheless, differences were observable when effects of heat and heavy metal stress were compared. Whereas heat stress preferentially activated the HSE promoter, heavy metals more strongly induced the HSPA1A promoter. We therefore analysed the HSPA1A promoter in more detail, by isolating and mutating the HSEs. The results indicate that the importance of the individual binding sites for HSF1 is determined by their sequence similarity to the consensus sequence and their position relative to the transcription start site, but they were not differentially affected by heat or heavy metal stress. In contrast, we found that other parts of the HSPA1A promoter have different impact on the response under different stress conditions. In this work we provide deeper insights into the regulation of HSP72 expression as a well as a method to quantitatively and sensitively evaluate different stressor on their potential to activate HSF1.

A Dominant Negative Antisense Approach Targeting ß-Catenin.

There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, ß-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of ß-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of ß-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of ß-catenin.

A sensitive, semi-quantitative mammalian two-hybrid assay.

Protein-protein interactions critically determine the function of a protein within the cell. Several methods have been developed for the analysis of protein interactions, including two-hybrid assays in yeast and mammals. Mammalian two-hybrid systems provide the ideal physiological environment to study the interactions of mammalian proteins; however, these approaches are limited in sensitivity and their ability to quantify interaction strength. Here, we present an inducible mammalian two-hybrid (iM2H) system using the small-molecule dimerizer rapalog for recruitment of multiple transactivation domains into the M2H system. This inducibility, combined with additional improvements of the iM2H components, results in an up to 100-fold increase in sensitivity compared with conventional M2H approaches. In addition, we include a number of reference interactions in our iM2H approach, which enable semiquantitative assessment of protein interactions. Using Groucho/Tle proteins and their binding partners, we demonstrate the applicability of our iM2H to established protein networks. Finally, to test the applicability of our system for drug screening, the interference of a small-molecule inhibitor on a known protein-protein interaction was tested, and the particular advantages of the internal reference interactions were shown.

An artificial HSE promoter for efficient and selective detection of heat shock pathway activity.

Detection of cellular stress is of major importance for the survival of cells. During evolution, a network of stress pathways developed, with the heat shock (HS) response playing a major role. The key transcription factor mediating HS signalling activity in mammalian cells is the HS factor HSF1. When activated it binds to the heat shock elements (HSE) in the promoters of target genes like heat shock protein (HSP) genes. They are induced by HSF1 but in addition they integrate multiple signals from different stress pathways. Here, we developed an artificial promoter consisting only of HSEs and therefore selectively reacting to HSF-mediated pathway activation. The promoter is highly inducible but has an extreme low basal level. Direct comparison with the HSPA1A promoter activity indicates that heat-dependent expression can be fully recapitulated by isolated HSEs in human cells. Using this sensitive reporter, we measured the HS response for different temperatures and exposure times. In particular, long heat induction times of 1 or 2 h were compared with short heat durations down to 1 min, conditions typical for burn injuries. We found similar responses to both long and short heat durations but at completely different temperatures. Exposure times of 2 h result in pathway activation at 41 to 44 °C, whereas heat pulses of 1 min lead to a maximum HS response between 47 and 50 °C. The results suggest that the HS response is initiated by a combination of temperature and exposure time but not by a certain threshold temperature.