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Toll-like receptor 4 (TLR4) are part of the innate immune system. They are capable of recognizing pathogen-associated molecular patterns (PAMPS) of microbes, and damage-associated molecular patterns (DAMPs) of damaged tissues. Activation of TLR4 initiates downstream signaling pathways that trigger the secretion of cytokines, type I interferons, and other pro-inflammatory mediators that are necessary for an immediate immune response. However, the systemic release of pro-inflammatory proteins is a powerful driver of acute and chronic inflammatory responses. Over the past decades, immense progress has been made in clarifying the molecular and regulatory mechanisms of TLR4 signaling in inflammation. However, the most common strategies used to study TLR4 signaling rely on genetic manipulation of the TLR4 or the treatment with agonists such as lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, which are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner. This review provides an overview of the structure, function and signaling pathways of the TLR4 and its fundamental role in endothelial cells under physiological and inflammatory conditions, as well as the advances in TLR4 modulation strategies.

Unlike traditional base materials such as concrete or masonry, there are no guidelines for rock as a base material for post-installed anchors. The varying rock properties (e.g., rock type, discontinuities) and numerous installation parameters (e.g., embedment depth, anchor diameter) leave engineers with limited information on design resistances, leading to an uncertain basis for anchor applications in rock. To identify the key parameters that determine rock as a base material, an evaluation of rock characteristics was conducted, combined with in situ pull-out tests in different key geologies (granite, limestone, mica schist, dolomite, granulite) and discrete element modeling, which has been found to be suitable for investigating the load-bearing behavior of post-installed anchors in rock. Discontinuities were identified as the main factor influencing the load-bearing capacity of post-installed anchors in rock mass. Based on the in situ investigations, assessment methods for rock as a base material were proposed, along with corresponding resistance partial safety factors for design of 2.5, 2.0, and 1.7 for high, medium, and low levels of uncertainty regarding possible inhomogeneities. A limit value R ≥ 36, associated with rebound hammer assessments, was defined for the low degree of uncertainty, showing limitations for schistose rock. This is concluded by a design approach for determining design resistances of shallow fasteners in rock mass. [ABSTRACT FROM AUTHOR]

The freeze-thaw behaviour of bonded fasteners in concrete is assessed according to the European Assessment Document 330499-01-0601 with freeze-thaw condition tests, which include 50 temperature cycles with a duration of 24 h between −20 °C and +20 °C on constantly loaded anchors. It is assumed that one cycle is equivalent to the temperature difference, which a bonded fastener undergoes in one year. Based on an analysis of a 28-year time series of air temperature data for Austria respecting the Alpine region, a modified test protocol with a temperature amplitude of 65 °C between −20 °C and +45 °C is compiled without a predefinition of the number of cycles, in order to simulate temperature differences that occur under real climatic conditions. The experimental test results obtained for both test procedures demonstrate that the stabilization of the displacements for the modified test series occurred after 185 temperature cycles, compared to the 50 cycles for the standard method. This means that an increase in the temperature amplitude of 25 °C in the higher temperature range leads to an approximately 3.5 times higher number of required temperature cycles until displacement stabilization is reached. It is concluded that the definition of the used temperature range for freeze-thaw testing in conjunction with climatic data should be critically considered, in order to possibly adapt pure freeze-thaw tests towards experiments that take into account real annual temperature differences.

Abstract Conifers often occur along steep gradients of diverse climates throughout their natural ranges, which is expected to result in spatially varying selection to local climate conditions. However, signals of climatic adaptation can often be confounded, because unraveled clines covary with signals caused by neutral evolutionary processes such as gene flow and genetic drift. Consequently, our understanding of how selection and gene flow have shaped phenotypic and genotypic differentiation in trees is still limited. A 40‐year‐old common garden experiment comprising 16 Douglas‐fir (Pseudotsuga menziesii) provenances from a north‐to‐south gradient of approx. 1,000 km was analyzed, and genomic information was obtained from exome capture, which resulted in an initial genomic dataset of >90,000 single nucleotide polymorphisms. We used a restrictive and conservative filtering approach, which permitted us to include only SNPs and individuals in environmental association analysis (EAA) that were free of potentially confounding effects (LD, relatedness among trees, heterozygosity deficiency, and deviations from Hardy–Weinberg proportions). We used four conceptually different genome scan methods based on FST outlier detection and gene–environment association in order to disentangle truly adaptive SNPs from neutral SNPs. We found that a relatively small proportion of the exome showed a truly adaptive signal (0.01%–0.17%) when population substructuring and multiple testing was accounted for. Nevertheless, the unraveled SNP candidates showed significant relationships with climate at provenance origins, which strongly suggests that they have featured adaptation in Douglas‐fir along a climatic gradient. Two SNPs were independently found by three of the employed algorithms, and one of them is in close proximity to an annotated gene involved in circadian clock control and photoperiodism as was similarly found in Populus balsamifera. Synthesis. We conclude that despite neutral evolutionary processes, phenotypic and genomic signals of adaptation to climate are responsible for differentiation, which in particular explain disparity between the well‐known coastal and interior varieties of Douglas‐fir.

Existing concrete specifications limit the crack width in order to fulfill the limits of serviceability and durability. The crack width calculation results differ depending on the influences of the variability of the used materials, the geometry, the actions and the uncertainty of the used calculation model itself. In this contribution, the influence of the variability of the input parameters on the calculated crack width is investigated using probabilistic methods in a real case study of a highly loaded tunnel lining cross‐section. The primary objective of this study is a sensitivity analysis to identify the geometric and material‐related input parameters with the most significant influence, which must be particularly controlled during execution for quality control purposes. The study in this article is based on the Eurocode deterministic model for calculating crack width for a stabilized cracking stage in late concrete age. [ABSTRACT FROM AUTHOR]

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization.

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies.

Toll-like receptor 4 (TLR4) are part of the innate immune system. They are capable of recognizing pathogen-associated molecular patterns (PAMPS) of microbes, and damage-associated molecular patterns (DAMPs) of damaged tissues. Activation of TLR4 initiates downstream signaling pathways that trigger the secretion of cytokines, type I interferons, and other pro-inflammatory mediators that are necessary for an immediate immune response. However, the systemic release of pro-inflammatory proteins is a powerful driver of acute and chronic inflammatory responses. Over the past decades, immense progress has been made in clarifying the molecular and regulatory mechanisms of TLR4 signaling in inflammation. However, the most common strategies used to study TLR4 signaling rely on genetic manipulation of the TLR4 or the treatment with agonists such as lipopolysaccharide (LPS) derived from the outer membrane of Gram-negative bacteria, which are often associated with the generation of irreversible phenotypes in the target cells or unintended cytotoxicity and signaling crosstalk due to off-target or pleiotropic effects. Here, optogenetics offers an alternative strategy to control and monitor cellular signaling in an unprecedented spatiotemporally precise, dose-dependent, and non-invasive manner. This review provides an overview of the structure, function and signaling pathways of the TLR4 and its fundamental role in endothelial cells under physiological and inflammatory conditions, as well as the advances in TLR4 modulation strategies. [ABSTRACT FROM AUTHOR]

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas.

While a certain level of inflammation is critical for humans to survive infection and injury, a prolonged inflammatory response can have fatal consequences. Pattern recognition Toll-like receptors (TLRs) are key players in the initiation of an inflammatory process. TLR2 is one of the most studied pattern recognition receptors (PRRs) and is known to form heterodimers with either TLR1, TLR4, TLR6, and TLR10, allowing it to recognize a wide range of pathogens. Although a large number of studies have been conducted over the past decades, there are still many unanswered questions regarding TLR2 mechanisms in health and disease. In this review, we provide an up-to-date overview of TLR2, including its homo- and heterodimers. Furthermore, we will discuss the pro- and anti-inflammatory properties of TLR2 and recent findings in prominent TLR2-associated infectious and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The interaction between monocytes and endothelial cells in inflammation is central to chemoattraction, adhesion, and transendothelial migration. Key players, such as selectins and their ligands, integrins, and other adhesion molecules, and their functions in these processes are well studied. Toll-like receptor 2 (TLR2), expressed in monocytes, is critical for sensing invading pathogens and initiating a rapid and effective immune response. However, the extended role of TLR2 in monocyte adhesion and migration has only been partially elucidated. To address this question, we performed several functional cell-based assays using monocyte-like wild type (WT), TLR2 knock-out (KO), and TLR2 knock-in (KI) THP-1 cells. We found that TLR2 promotes the faster and stronger adhesion of monocytes to the endothelium and a more intense endothelial barrier disruption after endothelial activation. In addition, we performed quantitative mass spectrometry, STRING protein analysis, and RT-qPCR, which not only revealed the association of TLR2 with specific integrins but also uncovered novel proteins affected by TLR2. In conclusion, we show that unstimulated TLR2 influences cell adhesion, endothelial barrier disruption, migration, and actin polymerization. [ABSTRACT FROM AUTHOR]

In endothelial cells (ECs), stimulation of Toll-like receptor 4 (TLR4) by the endotoxin lipopolysaccharide (LPS) induces the release of diverse pro-inflammatory mediators, beneficial in controlling bacterial infections. However, their systemic secretion is a main driver of sepsis and chronic inflammatory diseases. Since distinct and rapid induction of TLR4 signaling is difficult to achieve with LPS due to the specific and non-specific affinity to other surface molecules and receptors, we engineered new light-oxygen-voltage-sensing (LOV)-domain-based optogenetic endothelial cell lines (opto-TLR4-LOV LECs and opto-TLR4-LOV HUVECs) that allow fast, precise temporal, and reversible activation of TLR4 signaling pathways. Using quantitative mass-spectrometry, RT-qPCR, and Western blot analysis, we show that pro-inflammatory proteins were not only expressed differently, but also had a different time course when the cells were stimulated with light or LPS. Additional functional assays demonstrated that light induction promoted chemotaxis of THP-1 cells, disruption of the EC monolayer and transmigration. In contrast, ECs incorporating a truncated version of the TLR4 extracellular domain (opto-TLR4 ΔECD2-LOV LECs) revealed high basal activity with fast depletion of the cell signaling system upon illumination. We conclude that the established optogenetic cell lines are well suited to induce rapid and precise photoactivation of TLR4, allowing receptor-specific studies. [ABSTRACT FROM AUTHOR]

In long-lasting mass concrete structures the desired material properties of the concrete mix to realize a durable concrete and a concrete surface without cracks conflict with each other. The requirement of concrete with high durability leads to high thermal energy release and therefore, as another consequence, to high crack risk. Crack reduction is achieved by use of concrete with low hydration energy, which on the other hand leads to a decrease in concrete durability. Besides from optimized base materials and concrete technology, a gradient material distribution in the cross-section could reduce the problem since durable concrete is needed near the surface and the requirement of low-hydration energy is located in the center of the member. A simplified model is used to investigate the possible effect of a gradient concrete material distribution in mass concrete structures on crack reduction. The results of the analysis show that gradient concrete might contribute to lowering the constraint stresses and therefore the crack risk during concrete hardening.

Sensitivity analysis of backward concrete breakout when drilling through thin structural elements This technical paper deals with the backward damage of concrete when drilling through concrete structures of different thickness and composition. Particularly when installing fixings in very thin structural elements – such as slabs and panels of pre‐stressed hollow core elements or façades – it may be necessary to drill completely through the fastening surface when making the drill hole. Drilling through results in the occurrence of a backward concrete damage on the opposite side of the start of the drilling process. This is relevant if any post‐installed fastenings lose a large proportion of their embedment depth in the concrete due to the backward damage. In this contribution, based on more than 400 boreholes, the characteristics of the backward concrete damage are investigated as a function of the parameters component thickness, borehole diameter, maximum aggregate size, drilling method and impact energy of the drilling machine. Based on the investigations, it can be shown that the depths and volumes of the backward concrete damage follow a logarithmic normal distribution. This makes it possible to statistically describe the size of the backward damage and thus to find an approach for determining the remaining anchorage depth. [ABSTRACT FROM AUTHOR]

The freeze-thaw behaviour of bonded fasteners in concrete is assessed according to the European Assessment Document 330499-01-0601 with freeze-thaw condition tests, which include 50 temperature cycles with a duration of 24 h between −20 °C and +20 °C on constantly loaded anchors. It is assumed that one cycle is equivalent to the temperature difference, which a bonded fastener undergoes in one year. Based on an analysis of a 28-year time series of air temperature data for Austria respecting the Alpine region, a modified test protocol with a temperature amplitude of 65 °C between −20 °C and +45 °C is compiled without a predefinition of the number of cycles, in order to simulate temperature differences that occur under real climatic conditions. The experimental test results obtained for both test procedures demonstrate that the stabilization of the displacements for the modified test series occurred after 185 temperature cycles, compared to the 50 cycles for the standard method. This means that an increase in the temperature amplitude of 25 °C in the higher temperature range leads to an approximately 3.5 times higher number of required temperature cycles until displacement stabilization is reached. It is concluded that the definition of the used temperature range for freeze-thaw testing in conjunction with climatic data should be critically considered, in order to possibly adapt pure freeze-thaw tests towards experiments that take into account real annual temperature differences. [ABSTRACT FROM AUTHOR]

Conifers often occur along steep gradients of diverse climates throughout their natural ranges, which is expected to result in spatially varying selection to local climate conditions. However, signals of climatic adaptation can often be confounded, because unraveled clines covary with signals caused by neutral evolutionary processes such as gene flow and genetic drift. Consequently, our understanding of how selection and gene flow have shaped phenotypic and genotypic differentiation in trees is still limited.A 40‐year‐old common garden experiment comprising 16 Douglas‐fir (Pseudotsuga menziesii) provenances from a north‐to‐south gradient of approx. 1,000 km was analyzed, and genomic information was obtained from exome capture, which resulted in an initial genomic dataset of >90,000 single nucleotide polymorphisms. We used a restrictive and conservative filtering approach, which permitted us to include only SNPs and individuals in environmental association analysis (EAA) that were free of potentially confounding effects (LD, relatedness among trees, heterozygosity deficiency, and deviations from Hardy–Weinberg proportions). We used four conceptually different genome scan methods based on FST outlier detection and gene–environment association in order to disentangle truly adaptive SNPs from neutral SNPs.We found that a relatively small proportion of the exome showed a truly adaptive signal (0.01%–0.17%) when population substructuring and multiple testing was accounted for. Nevertheless, the unraveled SNP candidates showed significant relationships with climate at provenance origins, which strongly suggests that they have featured adaptation in Douglas‐fir along a climatic gradient. Two SNPs were independently found by three of the employed algorithms, and one of them is in close proximity to an annotated gene involved in circadian clock control and photoperiodism as was similarly found in Populus balsamifera. Synthesis. We conclude that despite neutral evolutionary processes, phenotypic and genomic signals of adaptation to climate are responsible for differentiation, which in particular explain disparity between the well‐known coastal and interior varieties of Douglas‐fir. [ABSTRACT FROM AUTHOR]

Influence of different load application mechanisms on displacement extrapolation of bonded fasteners The influence of three different load application mechanisms for sustained load testing on bonded fasteners is investigated in this contribution. These vary in terms of constancy of applied load and their readjustment during testing. However, all of them are permissible for sustained load tests on bonded fasteners. Since the applied load is directly connected to the measured displacements, which are extrapolated up to the desired design working life of 50 years, the influence of different load application mechanisms on the displacement prediction is investigated. An extended design service life is currently gaining more importance and therefore the estimation of possible influences during testing on the extrapolation is relevant. Consequently, obtained knowledge in terms of the effect of different load application mechanisms on the prediction could be helpful for the development of testing guidelines for an extended design working life of bonded fasteners. [ABSTRACT FROM AUTHOR]

Sustained load tests are necessary for product‐qualification of bonded fasteners. The aim of these sustained load tests is the evaluation of the fastening under constant tension load. Based on these tests, an extrapolation of the displacement behaviour concerning the assumed working life of the fastening is carried out. Three different measurands are of interest for sustained load tests, which are the failure load during the tension tests, the constancy of the applied sustained load, as well as the displacement. Due to the fact that every measurand is tainted with measurement uncertainty, the present paper aimed to evaluate the dimension of the expected measurement uncertainty, since this influences in consequence the forecast of the displacement behaviour over the working life of a bonded fastener. The assessment is done in accordance with the „Guide to the expression of Uncertainty in Measurement" for both, the maximum expected measurement uncertainty in compliance with the accuracy requirements of the relevant testing standards and with values from practice for the purpose of comparison. [ABSTRACT FROM AUTHOR]

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Abstract: Sustained load testing on bonded fasteners is used for the long‐term forecast of the fastener displacement behaviour. The application of the Findley law as extrapolation method is an intensively discussed topic. This method uses a power law to forecast the long‐term behaviour. As a result, also the measurement uncertainty from measured displacements and temperature effects are extrapolated over time. Currently sustained load tests for product qualification of bonded fasteners are regulated in the European Assessment Document EAD 330499, in which certain measurement intervals and the required accuracy are defined. Recent improvements in data acquisition lead to possible reductions of measurement intervals in order to make long‐term forecasts more precise. Therefore, measurement uncertainty of the displacement measurement gains importance. Considering this issue, the influence of the measurement device itself, the location of displacement measurement in the test setup and temperature changes are discussed within this contribution. [ABSTRACT FROM AUTHOR]

In long-lasting mass concrete structures the desired material properties of the concrete mix to realize a durable concrete and a concrete surface without cracks conflict with each other. The requirement of concrete with high durability leads to high thermal energy release and therefore, as another consequence, to high crack risk. Crack reduction is achieved by use of concrete with low hydration energy, which on the other hand leads to a decrease in concrete durability. Besides from optimized base materials and concrete technology, a gradient material distribution in the cross-section could reduce the problem since durable concrete is needed near the surface and the requirement of low-hydration energy is located in the center of the member. A simplified model is used to investigate the possible effect of a gradient concrete material distribution in mass concrete structures on crack reduction. The results of the analysis show that gradient concrete might contribute to lowering the constraint stresses and therefore the crack risk during concrete hardening. [ABSTRACT FROM AUTHOR]

Das dem derzeitigen Stand der Technik entsprechende Bemessungskonzept für Verankerungen führt bei randnahen Mehrfachbefestigungen unter Querbelastung und in weiterer Folge auch bei einer kombinierten Beanspruchung zu teilweise konservativen Ergebnissen. Aus bereits durchgeführten Untersuchungen zur Verbesserung des vorhandenen Bemessungskonzepts für randnahe Mehrfachbefestigungen unter Schrägzugbelastung geht hervor, dass solche Verankerungen nach dem Versagen der ersten Ankerreihe durch Betonkantenbruch noch Resthaltekräfte aufweisen, weshalb eine Berücksichtigung dieser zur Steigerung der Effizienz der Bemessung beitragen kann. Da die quantitative Beschaffenheit dieser Resttragfähigkeit jedoch noch weitgehend unerforscht ist, wird in diesem Beitrag auf Basis experimenteller Untersuchungen aufgezeigt, in welcher Größenordnung sich diese Resthaltekräfte bewegen. Hierzu wird eine randnahe Einzelbefestigung des Verbunddübeltyps zunächst unter reiner Querlast in Richtung des freien Bauteilrandes bis zum Eintritt des Betonkantenbruchs belastet und anschließend die zentrische Resttragfähigkeit bestimmt. Evaluation of the residual capacity in tension on bonded anchors close to the edge after concrete edge failure The state of the art design concept for anchorages in concrete leads to partly conservative results especially for fastenings close to the edge under shear load or subsequently under inclined load. Former investigations concerning the improvement of the actual design concept for anchorages close to the edge under inclined load show that such fastening situations have a residual capacity, although the first anchor row has already failed under shear load due to concrete edge failure. Therefore, the provision of this residual capacity can increase the performance of the design concept. Due to the lack in the quantitative range of the residual capacity, the scale of these forces was identified based on experimental tests within this article. Hence single bonded anchors close to the edge are loaded in a first step with a shear load towards the free edge until the concrete edge fails and afterwards the centric residual capacity is determined. [ABSTRACT FROM AUTHOR]

Background: Many northern-hemisphere forests are dominated by oaks. These species extend over diverse environmental conditions and are thus interesting models for studies of plant adaptation and speciation. The genomic toolbox is an important asset for exploring the functional variation associated with natural selection. Results: The assembly of previously available and newly developed long and short sequence reads for two sympatric oak species, Quercus robur and Quercus petraea, generated a comprehensive catalog of transcripts for oak. The functional annotation of 91 k contigs demonstrated the presence of a large proportion of plant genes in this unigene set. Comparisons with SwissProt accessions and five plant gene models revealed orthologous relationships, making it possible to decipher the evolution of the oak genome. In particular, it was possible to align 9.5 thousand oak coding sequences with the equivalent sequences on peach chromosomes. Finally, RNA-seq data shed new light on the gene networks underlying vegetative bud dormancy release, a key stage in development allowing plants to adapt their phenology to the environment. Conclusion: In addition to providing a vast array of expressed genes, this study generated essential information about oak genome evolution and the regulation of genes associated with vegetative bud phenology, an important adaptive traits in trees. This resource contributes to the annotation of the oak genome sequence and will provide support for forward genetics approaches aiming to link genotypes with adaptive phenotypes. [ABSTRACT FROM AUTHOR]

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy. [ABSTRACT FROM AUTHOR]

Augmented Toll-like receptor 4 (TLR4) expression was found in nearly 70% of patients with pancreatic adenocarcinoma, which is correlated with increased tumorigenesis and progression. In this study, we engineered a new light-oxygen-voltage-sensing (LOV) domain-based optogenetic cell line (opto-TLR4 PANC-1) that enables time-resolved activation of the NF-κB and extracellular-signal regulated kinases (ERK)1/2 signalling pathway upon blue light-sensitive homodimerisation of the TLR4-LOV fusion protein. Continuous stimulation with light indicated strong p65 and ERK1/2 phosphorylation even after 24 h, whereas brief light exposure peaked at 8 h and reached the ground level 24 h post-illumination. The cell line further allows a voltage-dependent TLR4 activation, which can be continuously monitored, turned on by light or off in the dark. Using this cell line, we performed different phenotypic cell-based assays with 2D and 3D cultures, with the aim of controlling cellular activity with spatial and temporal precision. Light exposure enhanced cell attachment, the formation and extension of invadopodia, and cell migration in 3D spheroid cultures, but no significant changes in proliferation or viability could be detected. We conclude that the opto-TLR4 PANC-1 cell line is an ideal tool for investigating the underlying molecular mechanisms of TLR4, thereby providing strategies for new therapeutic options. [ABSTRACT FROM AUTHOR]

Quercetin, a dietary flavonoid found in fruits and vegetables, has been described as a substance with many anti-cancer properties in a variety of preclinical investigations. In the present study, we demonstrate that 2D and 3D melanoma models exhibit not only different sensitivities to quercetin, but also opposite, cancer-promoting effects when metastatic melanoma spheroids are treated with quercetin. Higher concentrations of quercetin reduce melanoma growth in three tested cell lines, whereas low concentrations induce the opposite effect in metastatic melanoma spheroids but not in the non-metastatic cell line. High (>12.5 µM) or low (<6.3 µM) quercetin concentrations decrease or enhance cell viability, spheroid size, and cell proliferation, respectively. Additionally, melanoma cells cultivated in 2D already show significant caspase 3 activity at very low concentrations (>0.4 µM), whereas in 3D spheroids apoptotic cells, caspase 3 activity can only be detected in concentrations ≥12.5 µM. Further, we show that the tumor promoting or repressing effect in the 3D metastatic melanoma spheroids are likely to be elicited by a precisely controlled regulation of Nrf2/ARE-mediated cytoprotective genes, as well as ERK and NF-κB phosphorylation. According to the results obtained here, further studies are needed to better characterize the mechanisms of action underlying the pro- and anti-carcinogenic effects of quercetin on human melanomas. [ABSTRACT FROM AUTHOR]