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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.

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.

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.