Correlated fluorescence lifetime and spectral measurements in living cells

Studies of proteins' interaction in cells by FRET can take benefit from two important photo-physical properties describing fluorescent proteins: fluorescence emission spectrum and fluorescence lifetime. These properties provide specific and complementary information about the tagged proteins and their environment. However, none of them taken individually can completely quantify the involved fluorophore characteristics due to their multiparametric dependency with molecular environment, experimental conditions, and interpretation complexity. A solution to get a better understanding of the biological process implied at the cellular level is to combine the spectral and temporal fluorescence data acquired simultaneously at every cell region under investigation. We present the SLiM-SPRC160, an original temporal/spectral acquisition system for simultaneous lifetime measurements in 16 spectral channels directly attached to the descanned port of a confocal microscope with two-photon excitation. It features improved light throughput, enabling low-level excitation and minimum invasivity in living cells studies. To guarantee a fairly good level of accuracy and reproducibility in the measurements of fluorescence lifetime and spectra on living cells, we propose a rigorous protocol for running experiments with this new equipment that preserves cell viability. The usefulness of SLiM approach for the precise determination of overlapping fluorophores is illustrated with the study of known solutions of rhodamine. Then, we describe reliable FRET experiments in imaging mode realized in living cells using this protocol. We also demonstrate the benefit of localized fluorescence spectrum-lifetime acquisitions for the dynamic study of fluorescent proteins. proteins.     

Metallic Conductive Nanowires Elaborated by PVD Metal Deposition on Suspended DNA Bundles

Metallic conductive nanowires (NWs) with DNA bundle core are achieved, thanks to an original process relying on double‐stranded DNA alignment and physical vapor deposition (PVD) metallization steps involving a silicon substrate. First, bundles of DNA are suspended with a repeatable process between 2 µm high parallel electrodes with separating gaps ranging from 800 nm to 2 µm. The process consists in the drop deposition of a DNA lambda‐phage solution on the electrodes followed by a naturally evaporation step. The deposition process is controlled by the DNA concentration within the buffer solution, the drop volume, and the electrode hydrophobicity. The suspended bundles are finally metallized with various thicknesses of titanium and gold by a PVD e‐beam evaporation process. The achieved NWs have a width ranging from a few nanometers up to 100 nm. The electrical behavior of the achieved 60 and 80 nm width metallic NWs is shown to be Ohmic and their intrinsic resistance is estimated according to different geometrical models of the NW section area. For the 80 nm width NWs, a resistance of about few ohms is established, opening exploration fields for applications in microelectronics.     

New Beta-lactamases in Candidate Phyla Radiation: Owning Pleiotropic Enzymes Is a Smart Paradigm for Microorganisms with a Reduced Genome

The increased exploitation of microbial sequencing methods has shed light on the high diversity of new microorganisms named Candidate Phyla Radiation (CPR). CPR are mainly detected via 16S rRNA/metabarcoding analyses or metagenomics and are found to be abundant in all environments and present in different human microbiomes. These microbes, characterized by their symbiotic/epiparasitic lifestyle with bacteria, are directly exposed to competition with other microorganisms sharing the same ecological niche. Recently, a rich repertoire of enzymes with antibiotic resistance activity has been found in CPR genomes by using an in silico adapted screening strategy. This reservoir has shown a high prevalence of putative beta-lactamase-encoding genes. We expressed and purified five putative beta-lactamase sequences having the essential domains and functional motifs from class A and class B beta-lactamase. Their enzymatic activities were tested against various beta-lactam substrates using liquid chromatography-mass spectrometry (LC-MS) and showed some beta-lactamase activity even in the presence of a beta-lactamase inhibitor. In addition, ribonuclease activity was demonstrated against RNA that was not inhibited by sulbactam and EDTA. None of these proteins could degrade single- and double-stranded-DNA. This study is the first to express and test putative CPR beta-lactamase protein sequences in vitro. Our findings highlight that the reduced genomes of CPR members harbor sequences encoding for beta-lactamases known to be multifunction hydrolase enzymes.     

Risk-informed decision-making in the presence of epistemic uncertainty

An important issue in risk analysis is the distinction between epistemic and aleatory uncertainties. In this paper, the use of distinct representation formats for aleatory and epistemic uncertainties is advocated, the latter being modelled by sets of possible values. Modern uncertainty theories based on convex sets of probabilities are known to be instrumental for hybrid representations where aleatory and epistemic components of uncertainty remain distinct. Simple uncertainty representation techniques based on fuzzy intervals and p-boxes are used in practice. This paper outlines a risk analysis methodology from elicitation of knowledge about parameters to decision. It proposes an elicitation methodology where the chosen representation format depends on the nature and the amount of available information. Uncertainty propagation methods then blend Monte Carlo simulation and interval analysis techniques. Nevertheless, results provided by these techniques, often in terms of probability intervals, may be too complex to interpret for a decision-maker and we, therefore, propose to compute a unique indicator of the likelihood of risk, called confidence index. It explicitly accounts for the decision-maker's attitude in the face of ambiguity. This step takes place at the end of the risk analysis process, when no further collection of evidence is possible that might reduce the ambiguity due to epistemic uncertainty. This last feature stands in contrast with the Bayesian methodology, where epistemic uncertainties on input parameters are modelled by single subjective probabilities at the beginning of the risk analysis process.     

Design challenges of a 65 nm CMOS stacked folded differential PA structure for mobile communications

In this paper, a novel phase-locked loop (PLL) architecture with multiple charge pumps, which is used to design a fast-locking PLL and a low-phase-noise PLL, is proposed. The effective capacitance and resistance of the loop filter in terms of voltage is scaled up/down according to the locking status by controlling the magnitude and direction of the charge pump current. Two PLLs, one with a fast-locking characteristic and the other with a low-phase-noise characteristic, are designed and fabricated in a 0.35-μm CMOS process based on the proposed architecture. The fast-locking PLL has a locking time of less than 6 μs and a phase noise of −90.45 dBc/Hz at 1 MHz offset. The low-phase-noise PLL has a locking time of 25 μs, a phase noise of −105.37 dBc/Hz at 1 MHz offset, and a reference spur of −50 dBc. Both PLLs have an 851.2 MHz output frequency.     

Fault tolerant network design inspired by Physarum polycephalum

Physarum polycephalum, a true slime mould, is a primitive, unicellular organism that creates networks to transport nutrients while foraging. The design of these natural networks proved to be advanced, e.g. the slime mould was able to find the shortest path through a maze. The underlying principles of this design have been mathematically modelled in literature. As in real life the slime mould can design fault tolerant networks, its principles can be applied to the design of man-made networks. In this paper, an existing model and algorithm are adapted and extended with stimulation and migration mechanisms which encourage formation of alternative paths, optimize edge positioning and allow for automated design. The extended model can then be used to better design fault tolerant networks. The extended algorithm is applied to several national and international network configurations. Results show that the extensions allow the model to capture the fault tolerance requirements more accurately. The resulting extended algorithm overcomes weaknesses in geometric graph design and can be used to design fault tolerant networks such as telecommunication networks with varying fault tolerance requirements.     

A linear 60 GHz 65 nm-CMOS power amplifier realization and characterization for OFDM signal

A millimeter-wave Power Amplifier (PA) based on a 65nm CMOS technology from STMicroelectronics has been designed. The targeted feature is the unlicensed band around 60 GHz suitable for wireless personal area network application (WPAN). To optimize the linearity, the PA is designed under class A biasing to have an output compression point (OCP₁) close to its saturated Power (P _sat). S-parameters and large signal measurement results are demonstrated and compared with electromagnetic simulations. The PA offers a P _sat of 8.3 dBm, an OCP₁ of 6 dBm and a gain of 6.7 dB. The die area is 0.29 mm² with pads. Considering those results, one-tone simulations are not sufficient to characterize the linearity performances of the PA in its real conditions of use. Consequently, two-tone simulations are firstly performed. After, linearity figures of merit (FoM) are discussed applying an orthogonal frequency-division multiplexing (OFDM) modulated signal. The PA offers an adjacent channel power ratio (ACPR) of 15 dB and an error vector magnitude (EVM) of 20% at PA compression operating mode.     

Mechanistic investigation and selectivity of the grafting onto C60 of macroradicals prepared by cobalt-mediated radical polymerization

The grafting mechanism of poly(vinyl acetate) macroradicals prepared by cobalt-mediated radical polymerization onto C₆₀ is investigated. The experimental conditions directly impact the nature and stability of the PVAc/C₆₀ adducts. In the presence of residual initiating radicals that can compete with PVAc° macroradicals for addition onto C₆₀, mixtures of PVAc/C₆₀ adducts having between one and eight polymer chains per C₆₀ are formed. PVAc/C₆₀ adducts prepared with low [PVAc]:[C₆₀] ratios may contain weak C₆₀–C₆₀ bonds that further dissociate and account for the instability of the products. The formation of such dimers can be lessened by increasing the temperature from 30 °C to 100 °C. The temperature increase also allows a complete dissociation of the PVAc-Co dormant species into PVAc° macroradicals and an almost quantitative grafting of eight PVAc chains onto C₆₀, leading to well-defined C₆₀(PVAc)₈ octa-adducts. These results might shed new light on the grafting onto C₆₀ of macroradicals prepared by other CRP techniques.     

Reliability-based design optimization applied to structures submitted to random fatigue loads

Structural and Multidisciplinary Optimization - The reliability-based design optimization (RBDO) aims to find the most balanced design through a compromise between cost and safety when...