Vascular permeability and plasma leakage are immune-pathologies of severe dengue virus (DENV) infection, but the mechanisms underlying the exacerbated inflammation during DENV pathogenesis are ...unclear. Here, we demonstrate that TLR2, together with its co-receptors CD14 and TLR6, is an innate sensor of DENV particles inducing inflammatory cytokine expression and impairing vascular integrity in vitro. Blocking TLR2 prior to DENV infection in vitro abrogates NF-κB activation while CD14 and TLR6 block has a moderate effect. Moreover, TLR2 block prior to DENV infection of peripheral blood mononuclear cells prevents activation of human vascular endothelium, suggesting a potential role of the TLR2-responses in vascular integrity. TLR2 expression on CD14 + + classical monocytes isolated in an acute phase from DENV-infected pediatric patients correlates with severe disease development. Altogether, these data identify a role for TLR2 in DENV infection and provide insights into the complex interaction between the virus and innate receptors that may underlie disease pathogenesis.
Heterogeneity in mRNA Translation Sonneveld, Stijn; Verhagen, Bram M.P.; Tanenbaum, Marvin E.
Trends in cell biology,
08/2020, Volume:
30, Issue:
8
Journal Article
Peer reviewed
Open access
During mRNA translation, the genetic information stored in mRNA is translated into a protein sequence. It is imperative that the genetic information is translated with high precision. Surprisingly, ...however, recent experimental evidence has demonstrated that translation can be highly heterogeneous, even among different mRNA molecules derived from a single gene in an individual cell; multiple different polypeptides can be produced from a single mRNA molecule and the rate of translation can vary in both space and time. However, whether translational heterogeneity serves an important cellular function, or rather predominantly represents gene expression ‘noise’ remains an open question. In this review, we discuss the molecular basis and potential functions of such translational heterogeneity.
Different types of translational heterogeneity can be distinguished: cell or tissue heterogeneity (translation of a gene differs in distinct cell or tissue types), ‘intergenic’ heterogeneity (mRNAs derived from different genes are translated differentially), and ‘intragenic’ heterogeneity (different mRNAs derived from one gene in a single cell show translational heterogeneity).Recent studies using single-molecule imaging have revealed the widespread occurrence of intragenic translational heterogeneity.Intragenic translational heterogeneity can have multiple origins, including heterogeneity in primary mRNA sequence, RNA-binding proteins, RNA modifications, mRNA structure, and ribosome composition.Translational heterogeneity is likely to represent ‘noise’ as well as spatiotemporal regulation of translation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
mRNA translation is a key step in decoding genetic information. Genetic decoding is surprisingly heterogeneous because multiple distinct polypeptides can be synthesized from a single mRNA sequence. ...To study translational heterogeneity, we developed the MoonTag, a fluorescence labeling system to visualize translation of single mRNAs. When combined with the orthogonal SunTag system, the MoonTag enables dual readouts of translation, greatly expanding the possibilities to interrogate complex translational heterogeneity. By placing MoonTag and SunTag sequences in different translation reading frames, each driven by distinct translation start sites, start site selection of individual ribosomes can be visualized in real time. We find that start site selection is largely stochastic but that the probability of using a particular start site differs among mRNA molecules and can be dynamically regulated over time. This study provides key insights into translation start site selection heterogeneity and provides a powerful toolbox to visualize complex translation dynamics.
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•Development of MoonTag, a fluorescence labeling system to visualize translation•Combining MoonTag and SunTag enables visualization of translational heterogeneity•mRNAs from a single gene vary in initiation frequency at different start sites•Ribosomes take many different “paths” along the 5′ UTR of a single mRNA molecule
The MoonTag system is a fluorescence labeling system for visualizing translation of single mRNA molecules in live cells. Combining the MoonTag system with the orthogonal SunTag system enables simultaneous measurements of translation of two open reading frames in an mRNA and reveals that ribosomes differentially decode individual mRNA molecules.
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mRNA translation is a key step in gene expression. Proper regulation of translation efficiency ensures correct protein expression levels in the cell, which is essential to cell function. Different ...methods used to study translational control in the cell rely on population-based assays that do not provide information about translational heterogeneity between cells or between mRNAs of the same gene within a cell, and generally provide only a snapshot of translation. To study translational heterogeneity and measure translation dynamics, we have developed microscopy-based methods that enable visualization of translation of single mRNAs in live cells. These methods consist of a set of genetic tools, an imaging-based approach and sophisticated computational tools. Using the translation imaging method, one can investigate many new aspects of translation in single living cells, such as translation start-site selection, 3'-UTR (untranslated region) translation and translation-coupled mRNA decay. Here, we describe in detail how to perform such experiments, including reporter design, cell line generation, image acquisition and analysis. This protocol also provides a detailed description of the image analysis pipeline and computational modeling that will enable non-experts to correctly interpret fluorescence measurements. The protocol takes 2-4 d to complete (after cell lines expressing all required transgenes have been generated).
Nonsense-mediated decay (NMD) is a surveillance system that degrades mRNAs containing a premature termination codon (PTC) and plays important roles in protein homeostasis and disease. The efficiency ...of NMD is variable, impacting the clinical outcome of genetic mutations. However, limited resolution of bulk analyses has hampered the study of NMD efficiency. Here, we develop an assay to visualize NMD of individual mRNA molecules in real time. We find that NMD occurs with equal probability during each round of translation of an mRNA molecule. However, this probability is variable and depends on the exon sequence downstream of the PTC, the PTC-to-intron distance, and the number of introns both upstream and downstream of the PTC. Additionally, a subpopulation of mRNAs can escape NMD, further contributing to variation in NMD efficiency. Our study uncovers real-time dynamics of NMD, reveals key mechanisms that influence NMD efficiency, and provides a powerful method to study NMD.
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•Nonsense-mediated decay (NMD) of single mRNA molecules is visualized in live cells•Each terminating ribosome has an equal probability of inducing NMD•NMD efficiency is affected by the number and position of introns•Kinetics of XRN1-dependent decay of the 3′ mRNA cleavage fragment are uncovered
Hoek et al. develop a method to visualize nonsense-mediated mRNA decay (NMD) of individual mRNA molecules in live cells. Using this method, they uncover the precise timing of NMD, identify multiple parameters that affect NMD efficiency, and determine the kinetics of decay of the 3′ mRNA cleavage fragments by the exonuclease XRN1.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine ...hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers.
We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients.
Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community.
Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers.
EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
On the Shape of Surface Nanobubbles Borkent, Bram M; de Beer, Sissi; Mugele, Frieder ...
Langmuir,
01/2010, Volume:
26, Issue:
1
Journal Article
Peer reviewed
Open access
Previous AFM experiments on surface nanobubbles have suggested an anomalously large contact angle θ of the bubbles (typically ∼160° measured through the water) and a possible size dependence θ(R). ...Here we determine θ(R) for nanobubbles on smooth, highly oriented pyrolytic graphite (HOPG) with a variety of different cantilevers. It is found that θ(R) is constant within experimental error, down to bubbles as small as R = 20 nm, and is equal to 119 ± 4°. This result, which is the lowest contact angle for surface nanobubbles found so far, is very reproducible and independent of the cantilever type used, provided that the cantilever is clean and the HOPG surface is smooth. In contrast, we find that, for a particular set of cantilevers, the surface can become relatively rough because of precipitated matter from the cantilever onto the substrate, in which case larger nanoscopic contact angles (∼150°) show up. In addition, we address the issue of the set-point dependence. Once the set-point ratio is below roughly 95%, the obtained nanobubble shape changes and depends on both nanobubble size and cantilever properties (spring constant, material, and shape).
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IJS, KILJ, NUK, PNG, UL, UM
The unassembled components of the Khufu-1 ship, dated to the mid-3rd millennium BC were found in 1954 just south of the Khufu pyramid in Egypt. It has two mid-sheer planks, one on each side: 23 m ...long, and a maximum of 40 cm wide and 14 cm thick. These planks were made from the cedar of Lebanon (Cedrus libani) imported from Lebanon, 320 Nautical Miles (600 km) away, along the eastern coast of the Mediterranean. According to scholars who studied this ship, the ship planks, which were curved in three dimensions, to fit the hull, were produced from the cedar trunk by carving, not by bending. Given that cedar wood was imported and expensive in Egypt, the choice of the carving technique was unexpected, and above all, would yield a plank weaker than one twisted and bent from a straight timber. The two production techniques (carving and bending) were compared, and it was found that bending was superior, and in fact, was demonstrated to be the actual method of shaping the planks of the Khufu-1 ship.
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