The "flipping method" is a new straightforward way to both adsorb and organize microparticles at a liquid interface, with ultralow amounts of a surfactant and no other external forces than gravity. Here we demonstrate that it allows the adsorption of a variety of inorganic nanoparticles at an air/water interface, in an organized way, which is directly controlled by the surfactant concentration, ranging from amorphous to highly crystalline two-dimensional assemblies. With micromolar amounts of a conventional cationic surfactant (dodecyltrimethylammonium bromide, DTAB), nanoparticles of different compositions (silica, silver, and gold), sizes (down to 100 nm) and shapes (spheres and cubes) adsorb from the bulk and directly organize at the air/water interface, resulting in marked optical properties such as reflectivity or intense structural coloration.

Super-resolutive 3D tracking, such as PSF engineering or evanescent field imaging has long been used to track microparticles and to enhance the throughput of single molecules force spectroscopic measurements. However, current methods present two drawbacks. First, they lack precision compared with optical tweezers or AFM. Second, the dependence of their signal upon the position is complex creating the need for a time-consuming calibration step.
Here, we introduce a new optical technique that circumvents both issues and allows for a simple, versatile and efficient 3D tracking of diluted particles while offering a sub-nanometer frame-to-frame precision in all three spatial directions. The principle is to combine stereoscopy and interferometry, such that the z (axial) position is measured through the distance between two interferometric fringe patterns. The linearity of this stereoscopy technique alleviates the need for lookup tables while the structured interferometric pattern enhances precision. On the other hand, the extended spatial footprint of this PSF maximizes the number of photons detected per frame without the need of fancy cameras, nor the need for complex hardware. Hence, thanks to its simplicity and versatility, we believe that SDI (Stereo Darkfield Interferometry) technology has the potential to significantly enhance the spreading of 3D tracking.
We demonstrate the efficiency of this technique on various single-molecule measurements thanks to magnetic tweezers. In particular we demonstrate the precise quantification of two-state dynamics involving axial steps as short as 1 nm. We then show that SDI can be directly embedded in a commercial objective providing a means to track multiple single cells in 3D .

Bacterial proteins exported to the cell surface play key cellular functions. However, despite the interest to study the localisation of surface proteins such as adhesins, transporters or hydrolases, monitoring their dynamics in live imaging remains challenging, due to the limited availability of fluorescent probes remaining functional after secretion. In this work, we used the Escherichia coli intimin and the Listeria monocytogenes InlB invasin as surface exposed scaffolds fused with the recently developed chemogenetic fluorescent reporter protein FAST. Using both membrane permeant (HBR-3,5DM) and non-permeant (HBRAA-3E) fluorogens that fluoresce upon binding to FAST, we demonstrated that fully functional FAST can be exposed at the cell surface and used to specifically tag the external side of the bacterial envelop in both diderm and monoderm bacteria. Our work opens new avenues to study the organization and dynamics of the bacterial cell surface proteins.

Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.s.

Fluorescence-free micro-manipulation of nucleic acids (NA) allows the functional characterization of DNA/RNA processing proteins, without the interference of labels, but currently fails to detect and quantify their binding. To overcome this limitation, we developed a new method based on single-molecule force spectroscopy, called kinetic locking, that allows a direct in vitro visualization of protein binding while avoiding any kind of chemical disturbance of the protein’s natural function. We validate kinetic locking by measuring accurately the hybridization energy of ultrashort nucleotides (5,6,7 bases) and use it to measure the dynamical interactions of E. coli RecQ helicase with its DNA substrate.Competing Interest StatementThe authors have declared no competing interest.

Noninvasiveness, minimal handling, and immediate response are favorable features of fluorescence readout for high-throughput phenotyping of labeled plants.Yet, remote fluorescence imaging may suffer from an autofluorescent background and artificial or natural ambient light. In this work, the latter limitations are overcome by adopting reversibly photoswitchable fluorescent proteins (RSFPs) as labels and Speed OPIOM (out-of-phase imaging after optical modulation), a fluorescence imaging protocol exploiting dynamic contrast. Speed OPIOM can efficiently distinguish the RSFP signal from autofluorescence and other spectrally interfering fluorescent reporters like GFP. It can quantitatively assess gene expressions, even when they are weak. It is as quantitative, sensitive, and robust in dark and bright light conditions. Eventually, it can be used to nondestructively record abiotic stress responses like water or iron limitations in real time at the level of individual plants and even of specific organs. Such Speed OPIOM validation could find numerous applications to identify plant lines in selection programs, design plants as environmental sensors, or ecologically monitor transgenic plants in the environment.

Fluorescence has become a ubiquitous observable in biology. Yet it encounters limitations, which may originate from optical interferences such as ambient light, autofluorescence, and spectrally interfering fluorophores. In this review, we first report on dynamic contrast which can overcome these limitations. Then we specifically describe out-of-phase imaging after optical modulation, which proved relevant for multiplexed fluorescence imaging even under adverse optical conditions with several optical setups.

Interrogating living cells requires sensitive imaging of a large number of components in real time. The state-of-the-art of multiplexed imaging is usually limited to a few components. This review reports on the promise and the challenges of dynamic contrast to overcome this limitation.

Nucleus centering in mouse oocytes results from a gradient of actin-positive vesicle activity and is essential for developmental success. Here, we analyze 3D model simulations to demonstrate how a gradient in the persistence of actin-positive vesicles can center objects of different sizes. We test model predictions by tracking the transport of exogenous passive tracers. The gradient of activity induces a centering force, akin to an effective pressure gradient, leading to the centering of oil droplets with velocities comparable to nuclear ones. Simulations and experimental measurements show that passive particles subjected to the gradient exhibit biased diffusion toward the center. Strikingly, we observe that the centering mechanism is maintained in meiosis I despite chromosome movement in the opposite direction; thus, it can counteract a process that specifically off-centers the spindle. In conclusion, our findings reconcile how common molecular players can participate in the two opposing functions of chromosome centering versus off-centering.

The fast-developing field of synthetic biology enables broad applications of programmed microorganisms including the development of whole-cell biosensors, delivery vehicles for therapeutics, or diagnostic agents. However, the lack of spatial control required for localizing microbial functions could limit their use and induce their dilution leading to ineffective action or dissemination. To overcome this limitation, the integration of magnetic properties into living systems enables a contact-less and orthogonal method for spatiotemporal control. Here, we generated a magnetic-sensing Escherichia coli by driving the formation of iron-rich bodies into bacteria. We found that these bacteria could be spatially controlled by magnetic forces and sustained cell growth and division, by transmitting asymmetrically their magnetic properties to one daughter cell. We combined the spatial control of bacteria with genetically encoded-adhesion properties to achieve the magnetic capture of specific target bacteria as well as the spatial modulation of human cell invasions.

While precise tuning of gene expression levels is critical for most developmental pathways, the mechanisms by which the transcriptional output of dosage-sensitive molecules is established or modulated by the environment remain poorly understood. Here, we provide a mechanistic framework for how the conserved transcription factor BLMP-1/Blimp1 operates as a pioneer factor to decompact chromatin near its target loci hours before transcriptional activation and by doing so, regulates both the duration and amplitude of subsequent target gene transcription. This priming mechanism is genetically separable from the mechanisms that establish the timing of transcriptional induction and functions to canalize aspects of cell-fate specification, animal size regulation, and molting. A key feature of the BLMP-1-dependent transcriptional priming mechanism is that chromatin decompaction is initially established during embryogenesis and maintained throughout larval development by nutrient sensing. This anticipatory mechanism integrates transcriptional output with environmental conditions and is essential for resuming normal temporal patterning after animals exit nutrient-mediated developmental arrests.

Plasma-catalytic and thermo-catalytic methanation were assayed in the presence of a CeZrOx-supported Ni catalyst, proving that high CO2 conversions and high methane yields can be obtained under dielectric barrier discharge (DBD) plasma conditions and that they are maintained with time-on-stream over 100 h operating time. The characterization of the spent catalysts through TPD-MS, ATR-FTIR, TEM and HR-TEM and XRD evidenced the coexistence of a Ni0/NiO phase together with an increased presence of Ce3+ cations and oxygen vacancies, on the surface of the catalyst submitted to plasma catalytic operation. The different facts collected through physicochemical characterization point to our catalyst behaving like a PN junction, or like a fuel cell, with a P-side, the anode, i.e. the Ni-side releasing electrodes, while the CeZrOx support, N-side and cathode, acts as an acceptor. The DBD plasma, rich in ionic species and free electrodes, acts as the electrolyte, conducting the electrodes in the right direction. Oxygen accumulation on the surface of the catalyst during thermo-catalytic methanation leads to the formation of non-reactive adsorbed species, whereas Ni-sintering is favored. Under DBD plasma conditions, electron transfer is guaranteed and the adsorption–desorption of reactants and products is favored.

This study focuses on the use of a heterogeneous catalyst Ni/Ce0.58Zr0.42O2 to study the Sabatier reaction in conventional catalytic thermal heating and the dielectric barrier discharge plasma‐catalytic process. Its aim is to study the threshold temperature of the Sabatier reaction in plasma conditions. A set of experiments with different inlet flow rates is carried out in a plasma reactor to investigate the steady‐state temperature of the reaction. To estimate the threshold temperature of the Sabatier reaction more accurately, the temperature difference between the catalytic bed and the external surface of the reactor is calculated and simulated in COMSOL Multiphysics® software. Finally, the threshold temperature of the Sabatier reaction during plasma processing is assumed to be 116°C, based on the experimental data and simulation analysis.

A series of bi-metallic layered double hydroxide derived materials, containing a fixed amount of Ni promoted with various amounts of Fe were obtained by co-precipitation. The synthesized materials were characterized by X-ray diffraction (XRD), temperature-programmed reduction (H 2-TPR), temperature-programmed desorption of CO 2 (CO 2-TPD), elemental analysis and low temperature N 2 sorption and tested as catalysts in CO 2 methanation at atmospheric pressure. The obtained results confirmed the formation of mixed nano-oxides after thermal decomposition of the precursor and suggest successful introduction of both nickel and iron into the layers of Layered Double Hydroxides (LDHs). The introduction of Fe into the layered double hydroxides changed the interaction between Ni and supports matrix as proven by temperature programmed reduction (H 2-TPR). The introduction of low amount of iron influenced positively the catalytic activity in CO 2 methanation at 250 C, with CO 2 conversion increasing from 21% to 72% with CH 4 selec-tivity ranging from 97 to 99% at 250 C. No other products, except CH 4 and CO were registered during the experiments. In order to enhance the catalytic activity a non-thermal plasma created by dielectric barrier discharge was applied. The obtained results prove that * Corresponding author.

The research on soft actuators including liquid crystal elastomers (LCEs) becomes more and more appealing at a time when the expansion of artificial systems is blooming. Among the various LCE actuators, the bending deformation is often in the origin of many actuation modes. Here, a new strategy with plasma technology is developed to prepare single‐layer main‐chain LCEs with thermally actuated bending and contraction deformations. Two distinct reactions, plasma polymerization and plasma‐induced photopolymerization, are used to polymerize in one step the nematic monomer mixture aligned by magnetic field. The plasma polymerization forms cross‐linked but disoriented structures at the surface of the LCE film, while the plasma‐induced photopolymerization produces aligned LCE structure in the bulk. The actuation behaviors (bending and/or contraction) of LCE films can be adjusted by plasma power, reaction time, and sample thickness. Soft robots like crawling walker and flower mimic are built by LCE films with bending actuation.

Carbon-11 is undoubtedly an attractive PET radiolabeling synthon because carbon is present in all biological molecules. It is mainly found under 11CO2, but the latter being not very reactive, it is necessary to convert it into a secondary precursor. 11CO is an attractive precursor for labeling the carbonyl position through transition-metal mediated carbonylation because of its access to a wide range of functional groups (e.g., amides, ureas, ketones, esters, and carboxylic acids) present in most PET tracer molecules. However, the main limitations of 11CO labeling are the very short half-life of the radioisotope carbon-11 and its low concentration, and the low reactivity and poor solubility of 11CO in commonly used organic solvents. In this work, we show that a possible solution to these limitations is to use microfluidic reactor technology to perform carbonylation reactions, whilst a novel approach to generate CO from CO2 by plasma is described. The methodology consists of the decomposition of CO2 into CO by non-thermal DBD plasma at room temperature and atmospheric pressure, followed by the total incorporation of CO thus formed in the gas phase by carbonylation reaction, in less than 2 min of residence time. This “proof of principle” developed in carbon-12 would be further applied in carbon-11. Although considerable advances in 11CO chemistry have been reported in recent years, its application in PET tracer development is still an area of work in progress, because of the lack of commercially available synthesis instruments designed for 11C-carbonylations. To the best of our knowledge, such an innovative and efficient process, combining microfluidics and plasma, allowing the very fast organic synthesis of carbonyl molecules from CO2 with high yield, in mild conditions, has never been studied.

Although solid particles assembling on substrate surface is one of the key points for
developing membrane reactors, the technology of organizing nano/sub nanometer building
blocks into complex structures is still a challenge to scientists in years. In this work, amine
functional groups were deposited on the surface of different substrates via plasma enhanced
chemical vapor deposition (PECVD) technology and (3-aminopropyl)triethoxysilane
monomers were used as precursors. The influence of active gas, substrates, as well as
deposition time on the physico-chemical features of as-deposited film were investigated,
respectively. The highest density of amine of 5.5% on surface was obtained when Ar was
utilized as active gas and deposition time was 40 s. Furthermore, Y type zeolite particles at
sub-nano size were synthesized and subsequently used as a model material for testing the
immobilizing ability of plasma treated surface. The results clearly confirmed that a dense
mono or multi-layer of closely packed zeolite particles could be formed on the APTES as-
deposited surface after 24 hours’ immersion and the surface area of substrate could be
improved by the deposition of zeolite.

A miniaturized flow device has been developed to combine microfluidics technology and plasma process. In this microreactor, atmospheric pressure dielectric barrier discharges are generated in a gas in contact with a liquid phase. This study was conducted with plasma generated in ammonia in contact with a flow of liquid cyclohexane. Cyclohexylamine was synthesized with a good selectivity, and the process can be implemented to improve conversion and effectiveness. Numerical simulations confirmed that NH2 radicals are generated in the plasma and react with cyclohexyls radicals to achieve the reaction, giving a selectivity of 50% and a total molar conversion of 20% of cyclohexane. The effects of voltage and frequency on the selectivity and the experimental conversion rate were studied and discussed.

Metabolic alterations are a hallmark of the malignant transformation in cancer cells, which is characterized by multiple changes in metabolic pathways that are linked to macromolecule synthesis. This study aimed to explore whether salivary metabolites could help discriminate between breast cancer patients and healthy controls. Saliva samples from 23 breast cancer patients and 35 healthy controls were subjected to untargeted metabolomics using liquid chromatography-quadrupole time-of-flight mass spectrometry and a bioinformatics tool (XCMS Online), which revealed 534 compounds, characterized by their retention time in reverse-phase liquid chromatography and by the m/z ratio detected, that were shared by the two groups. Using the METLIN database, 31 compounds that were upregulated in the breast cancer group (p < 0.05) were identified, including seven oligopeptides and six glycerophospholipids (PG14:2, PA32:1, PS28:0, PS40:6, PI31:1, and PI38:7). In addition, pre-treatment and post-treatment saliva samples were analyzed for 10 patients who experienced at least a partial response to their treatment. In these patients, three peptides and PG14:2 were upregulated before but not after treatment. The area under the curve, sensitivity, and specificity for PG14:2 was 0.7329, 65.22%, and 77.14%, respectively. These results provide new information regarding the salivary metabolite profiles of breast cancer patients, which may be useful biomarkers. View Full-Text

The early detection of breast cancer enables the use of less aggressive treatment and increases patient survival. The transmembrane glycoprotein mucin 1, which is also known as cancer antigen 15‑3 (CA15‑3), is aberrantly glycosylated and overexpressed in a variety of epithelial cancers, and serves a crucial role in the progression of the disease. CA15‑3 is currently used as a marker of breast cancer. In the present study, CA15‑3 concentrations in saliva and blood of patients with breast cancer were evaluated to test new assays to detect salivary CA15‑3 in addition to ELISA and its diagnostic value. To the best of our knowledge, there are no previous reports of the use of chemiluminescence assay (CLIA) and electrochemiluminescence assay (ECLIA) in saliva. Saliva and blood were collected on the same day from patients with breast cancer (n=26) and healthy controls (n=28). For each subject, the level of serum CA15‑3 was measured using ECLIA, and the level of salivary CA15‑3 was measured using ECLIA, CLIA and enzyme‑linked immunosorbent assay (ELISA). ELISA and CLIA were able to detect CA15‑3 in saliva; however, ECLIA could not detect salivary CA15‑3. There was no significant difference between the mean serum and salivary CA15‑3 levels in patients with breast cancer or healthy controls. The levels of CA15‑3 were highest for luminal breast cancer subtypes and stage IV cases. A moderate correlation was observed between salivary and serum CA15‑3 levels as measured by ELISA in breast cancer patients (r=0.56; P=0.0047). The results demonstrated that ECLIA was not a good method to detect salivary CA15‑3, although it is the gold standard for detecting serum CA15‑3. The presence of CA15‑3 in saliva was confirmed, and this will be useful in future research. Further investigations are necessary to confirm the ability to detect salivary CA15‑3 and its correlation with serum CA15‑3.