BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures.

Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates.

Quantitative two-dimensional measurement of oil-film thickness by laser-induced fluorescence in a piston-ring model experiment.

This paper describes advances in using laser-induced fluorescence of dyes for imaging the thickness of oil films in a rotating ring tribometer with optical access, an experiment representing a sliding piston ring in an internal combustion engine. A method for quantitative imaging of the oil-film thickness is developed that overcomes the main challenge, the accurate calibration of the detected fluorescence signal for film thicknesses in the micrometer range. The influence of the background material and its surface roughness is examined, and a method for flat-field correction is introduced. Experiments in the tribometer show that the method yields quantitative, physically plausible results, visualizing features with submicrometer thickness.

Identification of multifaceted binding modes for pyrin and ASC pyrin domains gives insights into pyrin inflammasome assembly.

Inflammasomes are macromolecular complexes that mediate inflammatory and cell death responses to pathogens and cellular stress signals. Dysregulated inflammasome activation is associated with autoinflammatory syndromes and several common diseases. During inflammasome assembly, oligomerized cytosolic pattern recognition receptors recruit procaspase-1 and procaspase-8 via the adaptor protein ASC. Inflammasome assembly is mediated by pyrin domains (PYDs) and caspase recruitment domains, which are protein interaction domains of the death fold superfamily. However, the molecular details of their interactions are poorly understood. We have studied the interaction between ASC and pyrin PYDs that mediates ASC recruitment to the pyrin inflammasome, which is implicated in the pathogenesis of familial Mediterranean fever. We demonstrate that both the ASC and pyrin PYDs have multifaceted binding modes, involving three sites on pyrin PYD and two sites on ASC PYD. Molecular docking of pyrin-ASC PYD complexes showed that pyrin PYD can simultaneously interact with up to three ASC PYDs. Furthermore, ASC PYD can self-associate and interact with pyrin, consistent with previous reports that pyrin promotes ASC clustering to form a proinflammatory complex. Finally, the effects of familial Mediterranean fever-associated mutations, R42W and A89T, on structural and functional properties of pyrin PYD were investigated. The R42W mutation had a significant effect on structure and increased stability. Although the R42W mutant exhibited reduced interaction with ASC, it also bound less to the pyrin B-box domain responsible for autoinhibition and hence may be constitutively active. Our data give new insights into the binding modes of PYDs and inflammasome architecture.

TNF-α and IGF1 modify the microRNA signature in skeletal muscle cell differentiation.

BACKGROUND: Elevated levels of the inflammatory cytokine TNF-α are common in chronic diseases or inherited or degenerative muscle disorders and can lead to muscle wasting. By contrast, IGF1 has a growth promoting effect on skeletal muscle. The molecular mechanisms mediating the effect of TNF-α and IGF1 on muscle cell differentiation are not completely understood. Muscle cell proliferation and differentiation are regulated by microRNAs (miRNAs) which play a dominant role in this process. This study aims at elucidating how TNF-α or IGF1 regulate microRNA expression to affect myoblast differentiation and myotube formation. RESULTS: In this study, we analyzed the impact of TNF-α or IGF1 treatment on miRNA expression in myogenic cells. Results reveal that i) TNF-α and IGF1 regulate miRNA expression during skeletal muscle cell differentiation in vitro, ii) microRNA targets can mediate the negative effect of TNF-α on fusion capacity of skeletal myoblasts by targeting genes associated with axon guidance, MAPK signalling, focal adhesion, and neurotrophin signalling pathway, iii) inhibition of miR-155 in combination with overexpression of miR-503 partially abrogates the inhibitory effect of TNF-α on myotube formation, and iv) MAPK/ERK inhibition might participate in modulating the effect of TNF-α and IGF1 on miRNA abundance. CONCLUSIONS: The inhibitory effects of TNF-α or the growth promoting effects of IGF1 on skeletal muscle differentiation include the deregulation of known muscle-regulatory miRNAs as well as miRNAs which have not yet been associated with skeletal muscle differentiation or response to TNF-α or IGF1. This study indicates that miRNAs are mediators of the inhibitory effect of TNF-α on myoblast differentiation. We show that intervention at the miRNA level can ameliorate the negative effect of TNF-α by promoting myoblast differentiation. Moreover, we cautiously suggest that TNF-α or IGF1 modulate the miRNA biogenesis of some miRNAs via MAPK/ERK signalling. Finally, this study identifies indicative biomarkers of myoblast differentiation and cytokine influence and points to novel RNA targets.

Inhibition of neogenin dampens hepatic ischemia-reperfusion injury.

OBJECTIVE: Liver ischemia and reperfusion injury is a common source of significant morbidity and mortality following liver transplantation, hemorrhagic shock, or major hepatic surgery. Based on studies showing a critical role for the neuronal guidance receptor neogenin (Neo1) outside the nervous system in mediating tissue adaption during acute inflammation, we hypothesized that Neo1 enhances hepatic ischemia and reperfusion injury. DESIGN: Animal study. SETTING: University-based experimental laboratory. SUBJECTS: Wid-type, neogenin deficient and chimeric mice. INTERVENTIONS: Neogenin expression was evaluated during inflammatory stimulation in vitro and during ischemia and reperfusion injury in vivo, intravital microscopy performed to study intravascular flow characteristics. The extent of liver injury was evaluated using histology, serum levels of lactate dehydrogenase, aspartate, and alanine aminotransferase. The functional role of Neo1 during liver IR was evaluated in mice with gene targeted repression of neogenin (Neo1-/-), bone marrow chimeric animals and controls. In addition, functional inhibition of neogenin was performed using antibody injection. MEASUREMENTS AND MAIN RESULTS: We observed an induction of Neo1 during inflammation in vitro and ischemia and reperfusion in vivo. Intravital microscopy demonstrated a decreased ability of Neo1 leukocytes to attach to endothelial vascular wall during inflammation. Subsequent studies in Neo1 mice showed attenuated serum levels of lactate dehydrogenase, aspartate, alanine, and proinflammatory cytokines during hepatic ischemia and reperfusion injury. This was associated with improved hepatic histology scores. Studies in chimeric animals demonstrated that the hematopoietic Neo1 expression to be crucial for the observed results. Treatment with an anti-Neo1 antibody resulted in a significant reduction of experimental hepatic ischemia and reperfusion injury, involving attenuated variable of lactate dehydrogenase, alanine, aspartate, and cytokine levels. CONCLUSIONS: These data provide a unique role for Neo1 in the development of hepatic ischemia and reperfusion injury and identified Neo1 as a potential target to prevent liver dysfunction in the future.

Genome Sequence Analysis of Native Xenorhabdus Strains Isolated from Entomopathogenic Nematodes in Argentina.

Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.

The influence of bulk stoichiometry on near-ambient pressure reactivity of bare and Pt-loaded rutile TiO2(110).

The interaction of catalyst particles with reducible support materials can drastically change their reactivity. On rutile TiO2, processes like particle encapsulation (caused by the "strong metal-support interaction", SMSI) have long been known to depend on the initial reduction state of the oxide. Despite this knowledge, sample stoichiometry has rarely been controlled in a reproducible manner in the surface science literature. Here, we use scanning tunnelling microscopy (STM) to explore systematically how near-ambient pressures (0.1-1.0 mbar) of O2, H2, CO and CO2 affect blank and Pt-loaded rutile TiO2(110) surfaces of different bulk stoichiometry at 600 K. To this end, we present preparation recipes that result in a sample stoichiometry always converging back to the same value, which allows us to use the same samples with constant reduction state over hundreds of preparation cycles. Comparing a highly reduced and a near-stoichiometric TiO2 sample, we find that surface reactivity to all four gasses differs even without Pt loading. Most surprisingly, we find that the highly reduced TiO2(110) is oxidized by CO2, but this reaction is completely inhibited on the near-stoichiometric sample. Pt nanoparticles, in turn, become encapsulated after vacuum annealing on the reduced, but not on the near-stoichiometric sample. Encapsulation on the near-stoichiometric sample is achieved only after exposing it to 0.1 mbar H2 at 600 K. Interestingly, we also see a further modification of the already encapsulated particles on the reduced sample under the same conditions, such that they become embedded deeper in the TiO2(110) surface.

Evolution-inspired engineering of nonribosomal peptide synthetases.

Many clinically used drugs are derived from or inspired by bacterial natural products that often are produced through nonribosomal peptide synthetases (NRPSs), megasynthetases that activate and join individual amino acids in an assembly line fashion. In this work, we describe a detailed phylogenetic analysis of several bacterial NRPSs that led to the identification of yet undescribed recombination sites within the thiolation (T) domain that can be used for NRPS engineering. We then developed an evolution-inspired "eXchange Unit between T domains" (XUT) approach, which allows the assembly of NRPS fragments over a broad range of GC contents, protein similarities, and extender unit specificities, as demonstrated for the specific production of a proteasome inhibitor designed and assembled from five different NRPS fragments.

Schlieren measurements in the round cylinder of an optically accessible internal combustion engine.

This paper describes the design and experimental application of an optical system to perform schlieren measurements in the curved geometry of the cylinder of an optically accessible internal combustion engine. Key features of the system are a pair of cylindrical positive meniscus lenses, which keep the beam collimated while passing through the unmodified, thick-walled optical cylinder, and a pulsed, high-power light-emitting diode with narrow spectral width. In combination with a high-speed CMOS camera, the system is used to visualize the fuel jet after injection of hydrogen fuel directly into the cylinder from a high-pressure injector. Residual aberrations, which limit the system's sensitivity, are characterized experimentally and are compared to the predictions of ray-tracing software.

On-Surface Carbon Nitride Growth from Polymerization of 2,5,8-Triazido-s-heptazine.

Carbon nitrides have recently come into focus for photo- and thermal catalysis, both as support materials for metal nanoparticles as well as photocatalysts themselves. While many approaches for the synthesis of three-dimensional carbon nitride materials are available, only top-down approaches by exfoliation of powders lead to thin-film flakes of this inherently two-dimensional material. Here, we describe an in situ on-surface synthesis of monolayer 2D carbon nitride films as a first step toward precise combination with other 2D materials. Starting with a single monomer precursor, we show that 2,5,8-triazido-s-heptazine can be evaporated intact, deposited on a single crystalline Au(111) or graphite support, and activated via azide decomposition and subsequent coupling to form a covalent polyheptazine network. We demonstrate that the activation can occur in three pathways, via electrons (X-ray illumination), via photons (UV illumination), and thermally. Our work paves the way to coat materials with extended carbon nitride networks that are, as we show, stable under ambient conditions.

Does Cluster Encapsulation Inhibit Sintering? Stabilization of Size-Selected Pt Clusters on Fe3O4(001) by SMSI.

The metastability of supported metal nanoparticles limits their application in heterogeneous catalysis at elevated temperatures due to their tendency to sinter. One strategy to overcome these thermodynamic limits on reducible oxide supports is encapsulation via strong metal-support interaction (SMSI). While annealing-induced encapsulation is a well-explored phenomenon for extended nanoparticles, it is as yet unknown whether the same mechanisms hold for subnanometer clusters, where concomitant sintering and alloying might play a significant role. In this article, we explore the encapsulation and stability of size-selected Pt5, Pt10, and Pt19 clusters deposited on Fe3O4(001). In a multimodal approach using temperature-programmed desorption (TPD), X-ray photoelectron spectroscopy (XPS), and scanning tunneling microscopy (STM), we demonstrate that SMSI indeed leads to the formation of a defective, FeO-like conglomerate encapsulating the clusters. By stepwise annealing up to 1023 K, we observe the succession of encapsulation, cluster coalescence, and Ostwald ripening, resulting in square-shaped crystalline Pt particles, independent of the initial cluster size. The respective sintering onset temperatures scale with the cluster footprint and thus size. Remarkably, while small encapsulated clusters can still diffuse as a whole, atom detachment and thus Ostwald ripening are successfully suppressed up to 823 K, i.e., 200 K above the Hüttig temperature that indicates the thermodynamic stability limit.

Robust biclustering by sparse singular value decomposition incorporating stability selection.

MOTIVATION: Over the past decade, several biclustering approaches have been published in the field of gene expression data analysis. Despite of huge diversity regarding the mathematical concepts of the different biclustering methods, many of them can be related to the singular value decomposition (SVD). Recently, a sparse SVD approach (SSVD) has been proposed to reveal biclusters in gene expression data. In this article, we propose to incorporate stability selection to improve this method. Stability selection is a subsampling-based variable selection that allows to control Type I error rates. The here proposed S4VD algorithm incorporates this subsampling approach to find stable biclusters, and to estimate the selection probabilities of genes and samples to belong to the biclusters. RESULTS: So far, the S4VD method is the first biclustering approach that takes the cluster stability regarding perturbations of the data into account. Application of the S4VD algorithm to a lung cancer microarray dataset revealed biclusters that correspond to coregulated genes associated with cancer subtypes. Marker genes for different lung cancer subtypes showed high selection probabilities to belong to the corresponding biclusters. Moreover, the genes associated with the biclusters belong to significantly enriched cancer-related Gene Ontology categories. In a simulation study, the S4VD algorithm outperformed the SSVD algorithm and two other SVD-related biclustering methods in recovering artificial biclusters and in being robust to noisy data. AVAILABILITY: R-Code of the S4VD algorithm as well as a documentation can be found at http://s4vd.r-forge.r-project.org/.

In-cylinder temperature measurements via time-correlated single-photon counting of toluene laser-induced fluorescence through a fiber-based sensor.

In a near-production internal combustion engine, the effective fluorescence lifetime of toluene was determined by time-correlated single-photon counting with a minimally invasive fiber-optic spark-plug sensor. The lifetime measurement provided continuous crank-angle-resolved measurements of gas temperature. Proof-of-concept experiments in a motored four-cylinder spark-ignition engine were evaluated with a time resolution of 500 µs, yielding temperature precision of 25 K (standard deviation) at top-dead center. In these experiments, 10% toluene was added to the nonfluorescent base fuel iso-octane. Fluorescence lifetimes were related to temperature via calibration measurements in a high temperature pressure vessel, with the data fitted to a functional dependence derived from a previously published phenomenological model.

The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism.

RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.

Integrative Analysis of MicroRNA and mRNA Data Reveals an Orchestrated Function of MicroRNAs in Skeletal Myocyte Differentiation in Response to TNF-α or IGF1.

INTRODUCTION: Skeletal muscle cell differentiation is impaired by elevated levels of the inflammatory cytokine tumor necrosis factor-α (TNF-α) with pathological significance in chronic diseases or inherited muscle disorders. Insulin like growth factor-1 (IGF1) positively regulates muscle cell differentiation. Both, TNF-α and IGF1 affect gene and microRNA (miRNA) expression in this process. However, computational prediction of miRNA-mRNA relations is challenged by false positives and targets which might be irrelevant in the respective cellular transcriptome context. Thus, this study is focused on functional information about miRNA affected target transcripts by integrating miRNA and mRNA expression profiling data. METHODOLOGY/PRINCIPAL FINDINGS: Murine skeletal myocytes PMI28 were differentiated for 24 hours with concomitant TNF-α or IGF1 treatment. Both, mRNA and miRNA expression profiling was performed. The data-driven integration of target prediction and paired mRNA/miRNA expression profiling data revealed that i) the quantity of predicted miRNA-mRNA relations was reduced, ii) miRNA targets with a function in cell cycle and axon guidance were enriched, iii) differential regulation of anti-differentiation miR-155-5p and miR-29b-3p as well as pro-differentiation miR-335-3p, miR-335-5p, miR-322-3p, and miR-322-5p seemed to be of primary importance during skeletal myoblast differentiation compared to the other miRNAs, iv) the abundance of targets and affected biological processes was miRNA specific, and v) subsets of miRNAs may collectively regulate gene expression. CONCLUSIONS: Joint analysis of mRNA and miRNA profiling data increased the process-specificity and quality of predicted relations by statistically selecting miRNA-target interactions. Moreover, this study revealed miRNA-specific predominant biological implications in skeletal muscle cell differentiation and in response to TNF-α or IGF1 treatment. Furthermore, myoblast differentiation-associated miRNAs are suggested to collectively regulate gene clusters and targets associated with enriched specific gene ontology terms or pathways. Predicted miRNA functions of this study provide novel insights into defective regulation at the transcriptomic level during myocyte proliferation and differentiation due to inflammatory stimuli.

Profound effect of profiling platform and normalization strategy on detection of differentially expressed microRNAs--a comparative study.

BACKGROUND: Adequate normalization minimizes the effects of systematic technical variations and is a prerequisite for getting meaningful biological changes. However, there is inconsistency about miRNA normalization performances and recommendations. Thus, we investigated the impact of seven different normalization methods (reference gene index, global geometric mean, quantile, invariant selection, loess, loessM, and generalized procrustes analysis) on intra- and inter-platform performance of two distinct and commonly used miRNA profiling platforms. METHODOLOGY/PRINCIPAL FINDINGS: We included data from miRNA profiling analyses derived from a hybridization-based platform (Agilent Technologies) and an RT-qPCR platform (Applied Biosystems). Furthermore, we validated a subset of miRNAs by individual RT-qPCR assays. Our analyses incorporated data from the effect of differentiation and tumor necrosis factor alpha treatment on primary human skeletal muscle cells and a murine skeletal muscle cell line. Distinct normalization methods differed in their impact on (i) standard deviations, (ii) the area under the receiver operating characteristic (ROC) curve, (iii) the similarity of differential expression. Loess, loessM, and quantile analysis were most effective in minimizing standard deviations on the Agilent and TLDA platform. Moreover, loess, loessM, invariant selection and generalized procrustes analysis increased the area under the ROC curve, a measure for the statistical performance of a test. The Jaccard index revealed that inter-platform concordance of differential expression tended to be increased by loess, loessM, quantile, and GPA normalization of AGL and TLDA data as well as RGI normalization of TLDA data. CONCLUSIONS/SIGNIFICANCE: We recommend the application of loess, or loessM, and GPA normalization for miRNA Agilent arrays and qPCR cards as these normalization approaches showed to (i) effectively reduce standard deviations, (ii) increase sensitivity and accuracy of differential miRNA expression detection as well as (iii) increase inter-platform concordance. Results showed the successful adoption of loessM and generalized procrustes analysis to one-color miRNA profiling experiments.

Use of Rayleigh imaging and ray tracing to correct for beam-steering effects in turbulent flames.

Laser Rayleigh imaging has been applied in a number of flow and flame studies to measure concentration or temperature distributions. Rayleigh cross sections are dependent on the index of refraction of the scattering medium. The same index of refraction changes that provide contrast in Rayleigh images can also deflect the illuminating laser sheet. By applying a ray-tracing algorithm to the detected image, it is possible to correct for some of these beam-steering effects and thereby improve the accuracy of the measured field. Additionally, the quantification of the degree of beam steering through the flow provides information on the degradation of spatial resolution in the measurement. Application of the technique in a well-studied laboratory flame is presented, along with analysis of the effects of image noise and spatial resolution on the effectiveness of the algorithm.