Tethering beads to DNA offers a panel of single molecule techniques for the refined analysis of the conformational dynamics of DNA and the elucidation of the mechanisms of enzyme activity. Recent developments include the massive parallelization of these techniques achieved by the fabrication of dedicated nanoarrays by soft nanolithography. We focus here on two of these techniques: the Tethered Particle motion and Magnetic Tweezers allowing analysis of the behavior of individual DNA molecules in the absence of force and under the application of a force and/or a torque, respectively. We introduce the experimental protocols for the parallelization and discuss the benefits already gained, and to come, for these single molecule investigations.

The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.

The protective function of biological surfaces that are exposed to the exterior of living organisms is the result of a complex arrangement and interaction of cellular components. This is the case for the most external cornified layer of skin, the stratum corneum (SC). This layer is made of corneocytes, the elementary 'flat bricks' that are held together through adhesive junctions. Despite the well-known protective role of the SC under high mechanical stresses and rapid cell turnover, the subtleties regarding the adhesion and mechanical interaction among the individual corneocytes are still poorly known. Here, we explore the adhesion of single corneocytes at different depths of the SC, by pulling them using glass microcantilevers, and measuring their detachment forces. We measured their interplanar adhesion between SC layers, and their peripheral adhesion among cells within a SC layer. Both adhesions increased considerably with depth. At the SC surface, with respect to adhesion, the corneocyte population exhibited a strong heterogeneity, where detachment forces differed by more than one order of magnitude for corneocytes located side by side. The measured detachment forces indicated that in the upper-middle layers of SC, the peripheral adhesion was stronger than the interplanar one. We conclude that the stronger peripheral adhesion of corneocytes in the SC favors an efficient barrier which would be able to resist strong stresses.

All known eukaryotic topoisomerases are only able to relieve torsional stress in DNA. Nevertheless, it has been proposed that the introduction of positive DNA supercoiling is required for efficient sister-chromatid disjunction by Topoisomerase 2a during mitosis. Here we identify a eukaryotic enzymatic activity that introduces torsional stress into DNA. We show that the human Plk1-interacting checkpoint helicase (PICH) and Topoisomerase 3a proteins combine to create an extraordinarily high density of positive DNA supercoiling. This activity, which is analogous to that of a reverse-gyrase, is apparently driven by the ability of PICH to progressively extrude hypernegatively supercoiled DNA loops that are relaxed by Topoisomerase 3a. We propose that this positive supercoiling provides an optimal substrate for the rapid disjunction of sister centromeres by Topoisomerase 2a at the onset of anaphase in eukaryotic cells.

DEAD-box helicases are involved in all steps of RNA metabolism. They are ATP-dependent RNA binding proteins and RNA-dependent ATPases. They can displace short duplexes, but they lack processivity. Their mechanism and functioning are not clearly understood; classical or bulk biochemical assays are not sufficient to answer these questions. Single-molecule techniques provide useful tools, but they are limited in cases where the proteins are nonprocessive and give weak signals. We present here a new, magnetic-tweezers-based, single-molecule assay that is simple and that can sensitively measure the displacement time of a small, hybridized, RNA oligonucleotide. Tens of molecules can be analyzed at the same time. Comparing the displacement times with and without a helicase gives insights into the enzymatic activity of the protein. We used this assay to study yeast Ded1, which is orthologous to human DDX3. Although Ded1 acts on a variety of substrates, we find that Ded1 requires an RNA substrate for its ATP-dependent unwinding activity and that ATP hydrolysis is needed to see this activity. Further, we find that only intramolecular single-stranded RNA extensions enhance this activity. We propose a model where ATP-bound Ded1 stabilizes partially unwound duplexes and where multiple binding events may be needed to see displacement.

The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies

The biological processes associated with the onset of schizophrenia remain largely unknown. Current hypotheses favor gene × environment interactions as supported by our recent report about DNA methylation changes during the onset of psychosis. Here, we conducted the first longitudinal transcriptomic analysis of blood samples from 31 at-risk individuals who later converted to psychosis and 63 at-risk individuals who did not. Individuals were followed for a maximum of 1 year. Blood samples were collected at baseline and at the end of follow-up and individuals served as their own controls. Differentially expressed genes between the 2 groups were identified using the RNA sequencing of an initial discovery subgroup (n = 15 individuals). The most promising results were replicated using high-throughput real-time qPCR in the whole cohort (n = 94 individuals). We identified longitudinal changes in 4 brain-expressed genes based on RNAseq analysis. One of these genes (CPT1A) was replicated in the whole cohort. The previously observed hypermethylation in NRP1 and GSTM5 during the onset of psychosis correlated with a decrease in corresponding gene expression. RNA sequencing also identified 2 co-expression networks that were impaired after conversion compared with baseline-the Wnt pathway including AKT1, CPT1A and semaphorins, and the Toll-like receptor pathway, related to innate immunity. This longitudinal study of transcriptomic changes in individuals with at-risk mental state revealed alterations during conversion to psychosis in pathways and genes relevant to schizophrenia. These results may be a first step toward better understanding psychosis onset. They may also help to identify new biomarkers and targets for disease-modifying therapeutic strategies

The use of light to control the expression of genes and the activity of proteins is a rapidly expanding field. While many of these approaches use a fusion between a light activatable protein and the protein of interest to control the activity of the latter, it is also possible to control the activity of a protein by uncaging a specific ligand. In that context, controlling the activation of a protein fused to the modified estrogen receptor (ERT) by uncaging its ligand cyclofen-OH has emerged as a generic and versatile method to control the activation of proteins quantitatively, quickly and locally in a live organism. Here, we present the experimental details behind this approach.

Glutamate is a major excitatory neurotransmitter, and impaired glutamate clearance following synaptic release promotes spillover, inducing extra-synaptic signaling. The effects of glutamate spillover on animal behavior and its neural correlates are poorly understood. We developed a glutamate spillover model in Caenorhabditis elegans by inactivating the conserved glial glutamate transporter GLT-1. GLT-1 loss drives aberrant repetitive locomotory reversal behavior through uncontrolled oscillatory release of glutamate onto AVA, a major interneuron governing reversals. Repetitive glutamate release and reversal behavior require the glutamate receptor MGL-2/mGluR5, expressed in RIM and other interneurons presynaptic to AVA. mgl-2 loss blocks oscillations and repetitive behavior; while RIM activation is sufficient to induce repetitive reversals in glt-1 mutants. Repetitive AVA firing and reversals require EGL-30/Gαq, an mGluR5 effector. Our studies reveal that cyclic autocrine presynaptic activation drives repetitive reversals following glutamate spillover. That mammalian GLT1 and mGluR5 are implicated in pathological motor repetition suggests a common mechanism controlling repetitive behaviors.

Stochastic mechanisms diversify cell fate in organisms ranging from bacteria to humans [1, 2, 3, 4]. In the anchor cell/ventral uterine precursor cell (AC/VU) fate decision during C. elegans gonadogenesis, two “α cells,” each with equal potential to be an AC or a VU, interact via LIN-12/Notch and its ligand LAG-2/DSL [5, 6]. This LIN-12/Notch-mediated interaction engages feedback mechanisms that amplify a stochastic initial difference between the two α cells, ensuring that the cell with higher lin-12 activity becomes the VU while the other becomes the AC [7, 8, 9]. The initial difference between the α cells was originally envisaged as a random imbalance from “noise” in lin-12 expression/activity [6]. However, subsequent evidence that the relative birth order of the α cells biases their fates suggested other factors may be operating [7]. Here, we investigate the nature of the initial difference using high-throughput lineage analysis [10]; GFP-tagged endogenous LIN-12, LAG-2, and HLH-2, a conserved transcription factor that orchestrates AC/VU development [7, 11]; and tissue-specific hlh-2 null alleles. We identify two stochastic elements: relative birth order, which largely originates at the beginning of the somatic gonad lineage three generations earlier, and onset of HLH-2 expression, such that the α cell whose parent expressed HLH-2 first is biased toward the VU fate. We find that these elements are interrelated, because initiation of HLH-2 expression is linked to the birth of the parent cell. Finally, we provide a potential deterministic mechanism for the HLH-2 expression bias by showing that hlh-2 is required for LIN-12 expression in the α cells.

In addition to the search for new Thermal Barrier Coating (TBCs) systems with increased reliability over very long time periods, the repair of current systems is a technological and economic issue for both civilian and military engine end-users. This paper describes the latest version of the deposition process known as the Low-Power Plasma Reactor (LPPR) process, specially developed to repair locally damaged TBCs. The LPPR process enables micro/nanostructured TBCs to be made from nitrate salts in aqueous solutions, which are sprayed in an Ar/O2 plasma discharge at low power (240 W) and transformed into oxide coatings. A new injection device was designed to produce a fairly homogenous and reproducible spray to repair partially spalled APS and EB-PVD TBCs deposited on small flat coupons. The microstructure and the stability of the LPPR TBCs were assessed, in particular using SEM observations, during ageing tests under various time/temperature conditions. The Particle Image Velocimetry (PIV) technique and associated modeling have proved that the nitrates impact the substrate in a liquid state even in the presence of plasma and a vacuum. Due to the liquid state of the precursors, the new LPPR TBC seals the damaged areas and deeply infiltrates all porosities and failure cracks in the original coatings. This research has enabled the new version of the LPPR process to be validated as a simple, efficient, cheap and promising way to repair locally damaged TBCs

The present study focuses on the hybrid plasma catalytic process for CO2 methanation. This plasma-catalytic process, based on the combination of a DBD plasma and Ni/CeZrO2 catalyst, has several advantages over conventional catalysis: it operates at ambient conditions and requires no external heating. An optimization of the process considering the effect of the different operational parameters such as voltage, GHSV, catalyst mass, flow rate, discharge length, is herein presented. Moreover, a spectroscopic study, aiming to understand the mechanism of the reaction, is also showed. At temperatures around 270 °C and under adiabatic conditions, CO2 conversion rates of about 80% were measured, with a CH4 selectivity greater than 95%.

Lab‐On‐A‐Chips promise solutions for high throughput and specific analysis for environmental and health applications, with the challenge to develop materials allowing fast, easy, and cheap microfabrication and efficient surface treatment. Cyclic olefin copolymer (COC) is a promising thermoplastic, easily microfabricated for both rapid prototyping and low‐cost mass production of microfluidic devices but still needing efficient surface modification strategies. This study reports for the first time the optimization of an easy COC silica coating process by plasma‐enhanced chemical vapor deposition at atmospheric pressure with plasma jet and tetraethylorthosilicate as precursor, leading to a 158 ± 7 nm thickness and a 14‐day‐stability of hydrophilic properties for a COC‐embedded microchannel (100 µm), paving the way for a simplified and controlled COC surface modification.

Performance and mechanism of non-thermal plasma (NTP) technology in removing glucocorticoids (GCs) was investigated using a dielectric barrier discharge (DBD) reactor with fluocinolone acetonide (FA), triamcinolone acetonide (TA) and clobetasol propionate (CP) as representative compounds. Effects of discharge power, plasma-working gases, initial pH, coexistence of ions, and various water matrices (ultrapure water, lake water, drinking water, wastewater effluent) on GC removal and energy yield were evaluated. The results confirm that DBD treatment could efficiently remove FA, TA, and CP, achieving efficiency of 72% (k = 0.0126 min(-1)), 71% (k = 0.0096 min(-1)), and 74% (k = 0.0116 min(-1)), respectively in air-DBD system at 45.2 W, with the process following the first order kinetics and energy yield of 6 mg kW(-1) h(-1). The removal efficiency decreased when adding radical scavengers, indicating that hydroxyl radicals played an important role in GC degradation, while other active species (such as solvated electrons (e(aq)(-)), ozone (O-3), hydrogen peroxide (H2O2) and ultraviolet photolysis (UV)) also contribute to GC degradation. The intermediates generated during the process were analyzed using quadrupole time-of-flight mass spectrometry (QTOF-MS). A total of 23 transformation products of FA, TA and CP were identified, and it was noted that substitution of halogen atoms with center dot OH, oxidation of hydroxyl group to keto acid, decarboxylation of the keto acid, addition of center dot OH, intramolecular cyclization, and hydrolysis of esters occurred during GC degradation by DBD treatment.

The assembly of nano- and micro-scale building blocks on surface has been the focus of intense interest in materials science for years. In this work, (3-aminopropyl)triethoxysilane (APTES) carrying one primary amino group was deposited on various substrate surfaces using the plasma polymerization method. The key plasma parameters i.e. pressure and power were varied to obtained the highest density of primary amino groups. The influence of such parameters on the characteristics of deposited layers (e.g. chemical structure, adhesion strength, growth rate, etc.) was systemically investigated using various characterization methods such as XPS, FTIR, ellipsometry and so on. Meanwhile, three types of particles (AuNPs, zeolites and gold@zeolites) with sizes from nano- to submicro-range were synthesized and further used as model building blocks. Subsequently, the prepared particles were deposited onto cyclic olefin copolymer (COC) substrate surfaces, which were pre-functionalized by deposition of the plasma polymer layer using the parameters of pressure = 1.0 mbar and power = 30 W. The results confirmed the formation of membrane structures consisting of highly packed particles on the COC surface, and such immobilized structures showed high stability against flowing water, evidencing the good immobilization ability of deposited APTES layers with amino groups.

Nematic liquid crystal elastomer (LCE) micropillars with reversible thermomechanical deformations and photo luminescence (PL) intensity variations were successfully fabricated by introducing a cyano-oligo(p-phenylene vinylene) dye as a chemical crosslinker. The PL intensity of the micropillars decreased and increased reversibly during the thermal-deformation process. We studied in detail the possible factors that influence the PL intensity variations of the micropillars, including temperature variation, contraction/extension and phase transition. The dye molecules mainly kept the “monomer” state in the micropillars during the thermo-activated deformation. It was found that the phase transition from nematic to isotropic of the LCEs played the major role in the PL intensity variations. This kind of micropillar may have potential application in fluorescent soft sensors and actuators.

In the present work, gold based catalysts were synthesized and immobilized on the surface of cyclic olefin copolymer (COC) microreactors. The microreactors were subsequently applied in a homemade microfluidic system for synthesizing benzaldehyde by oxidation of benzyl alcohol in water medium. The Au nanoparticles (NPs) immobilized on the inner surface of the microchannel showed a very high selectivity (94%) for benzaldehyde, while zeolite NPs exhibited only an adsorption feature to this reaction. Moreover, the results showed that the AuNP catalytic activity was maintained for at least 9 hours. However, the obtained conversion with AuNPs was only 20%, indicating a relatively low productivity. In comparison, AuNPs assembled on the surface of zeolite NPs (AuNPs@zeolite) and immobilized in the microchannel showed the best catalytic performance, as the highest benzaldehyde selectivity (>99%) with a relatively high benzyl alcohol conversion of 42.4% was achieved under the same conditions. To the best of our knowledge, this is the first example demonstrating the use of AuNP or AuNP@zeolite catalysts in a microsystem performing such high selectivity for benzaldehyde in water medium.

Photoredox/nickel dual catalysis using easily oxidized bis-catecholato hypercoordinated silicon derivatives as radical sources and acyl chlorides as electrophiles allows a new method of formation of dialkyl and alkyl-aryl ketones as well as dibenzyl ketones which are less easily accessed. Flow chemistry can be used.

Modified UNIFAC (Dortmund) parameters for the interactions of the amino group at cycloaliphatic hydrocarbon with the hydroxyl group or the methanol group were determined by means of literature and our own experimental data. The vapor–liquid equilibria of the three binary systems cyclohexylamine + 2-butanol, cyclohexylamine + 2-methyl-2-butanol, and cyclohexylamine + 1-hexanol were measured isothermally with the dynamic method. The predictions of phase equilibrium data using the new parameter set for the amino group at cycloaliphatic hydrocarbon are compared to the predictions using the amino group at aliphatic hydrocarbon. The improvement of the description of experimental data is shown for binary and ternary systems.

Immobilization of gold nanoparticles (AuNPs) on the surface of zeolite has received a great interest due to Au@zeolite’s unique characteristics and high performance for catalysis. In this work we studied the grafting of two different functional molecules; one having an amine group (3-aminopropyl)triethoxysilane (APTES) and the second having a thiol group (3-mercaptopropyl)trimethoxysilane (MPTES) on the surface of zeolite using the same wet chemistry method. The modified zeolite surfaces were characterized using zeta potential measurements; diffuse reflectance infrared fourier transform (DRIFT) and X-ray photoelectron spectroscopy (XPS). The results confirmed a successful deposition of both functional groups at the topmost surface of the zeolite. Furthermore; transmission electron microscopy (TEM), ultraviolet-visible (UV-Vis) spectroscopy and XPS results clearly evidenced that APTES provided a better AuNPs immobilization than MPTES as a result of; (1) less active functions obtained after MPTES deposition, and (2) the better attaching ability of thiol to the gold surface.