Self-programmed enzyme phase separation and multiphase coacervate droplet organization

Membraneless organelles are phase-separated droplets that are dynamically assembled and dissolved in response to biochemical reactions in cells. Complex coacervate droplets produced by associative liquid–liquid phase separation offer a promising approach to mimic such dynamic compartmentalization. Here, we present a model for membraneless organelles based on enzyme/polyelectrolyte complex coacervates able to induce their own condensation and dissolution. We show that glucose oxidase forms coacervate droplets with a cationic polysaccharide on a narrow pH range, so that enzyme-driven monotonic pH changes regulate the emergence, growth, decay and dissolution of the droplets depending on the substrate concentration. Significantly, we demonstrate that time-programmed coacervate assembly and dissolution can be achieved in a single-enzyme system. We further exploit this self-driven enzyme phase separation to produce multiphase droplets via dynamic polyion self-sorting in the presence of a secondary coacervate phase. Taken together, our results open perspectives for the realization of programmable synthetic membraneless organelles based on self-regulated enzyme/polyelectrolyte complex coacervation.

Self-programmed enzyme phase separation is exploited to assemble dynamic multiphase coacervate droplets via spontaneous polyion self-sorting under non-equilibrium conditions.     

Quantum dot-based multiplexed fluorescence resonance energy transfer

We demonstrate the use of luminescent quantum dots (QDs) conjugated to dye-labeled protein acceptors for nonradiative energy transfer in a multiplexed format. Two configurations were explored: (1) a single color QD interacting with multiple distinct acceptors and (2) multiple donor populations interacting with one type of acceptor. In both cases, we showed that simultaneous energy transfer between donors and proximal acceptors can be measured. However, data analysis was simpler for the configuration where multiple QD donors are used in conjunction with one acceptor. Steady-state fluorescence results were corroborated by time-resolved measurements where selective shortening of QD lifetime was measured only for populations that were selectively engaged in nonradiative energy transfer.     

ACTION DES HYDRAZINES ET DE L'HYDROXYLAMINE SUR QUELQUES PHOSPHONATES β, β-BIFONCTIONNALISES: SYNTHESE DE PHOSPHONOAMINOPYRAZOLES ET ISOXAZOLES

Abstract

Reaction of hydrazines and hydroxylamine with β, β-bifunctionalized phosphonates 1 and 1′ leads to phosphoaminopyrazoles and isoxazoles 2 3, 4 and 5. The structure of all obtained products was confirmed by NMR and IR spectroscopy.     

Spatiotemporal multicolor labeling of individual cells using peptide-functionalized quantum dots and mixed delivery techniques

Multicolor fluorescent labeling of both intra- and extracellular structures is a powerful technique for simultaneous monitoring of multiple complex biochemical processes. This approach remains extremely challenging, however, as it often necessitates the combinatorial use of numerous targeting probes (e.g., antibodies), multistep bioconjugation chemistries, different delivery strategies (e.g., electroporation or transfection reagents), cellular fixation coupled with membrane permeabilization, and complex spectral deconvolution. Here, we present a nanoparticle-based fluorescence labeling strategy for the multicolor labeling of distinct subcellular compartments within live cells without the need for antibody conjugation or cellular fixation/permeabilization. This multipronged approach incorporates an array of delivery strategies, which localize semiconductor quantum dots (QDs) to various subcellular structures. QD uptake is implemented in a spaciotemporal manner by staggering the delivery of QD-peptide composites and exploiting various innate (peptide-mediated endocytosis, peptide-membrane interaction, polymer-based transfection) along with physical (microinjection) cellular delivery modalities to live cells growing in culture over a 4 day period. Imaging of the different intracellular labels is simplified by the unique photophysical characteristics of the QDs in combination with Fo?rster resonance energy transfer sensitization, which allow for multiple spectral windows to be accessed with one excitation wavelength. Using this overall approach, QDs were targeted to both early and late endosomes, the cellular cytosol, and the plasma membrane in live cells, ultimately allowing for simultaneous five-color fluorescent imaging.     

Enhancing the stability and biological functionalities of quantum dots via compact multifunctional ligands

We have designed and synthesized a series of modular ligands based on poly(ethylene glycol) (PEG) coupled with functional terminal groups to promote water-solubility and biocompatibility of quantum dots (QDs). Each ligand is comprised of three modules: a PEG single chain to promote hydrophilicity, a dihydrolipoic acid (DHLA) unit connected to one end of the PEG chain for strong anchoring onto the QD surface, and a potential biological functional group (biotin, carboxyl, and amine) at the other end of the PEG. Water-soluble QDs capped with these functional ligands were prepared via cap exchange with the native hydrophobic caps. Homogeneous QD solutions that are stable over extended periods of time and over a broad pH range were prepared. Surface binding assays and cellular internalization and imaging showed that QDs capped with DHLA-PEG-biotin strongly interacted with either NeutrAvidin immobilized on surfaces or streptavidin coupled to proteins which were subsequently taken up by live cells. EDC coupling in aqueous buffer solutions was also demonstrated using resonance energy transfer between DHLA-PEG-COOH-functionalized QDs and an amine-terminated dye. The new functional surface ligands described here provide not only stable and highly water-soluble QDs but also simple and easy access to various biological entities.     

Complex impedance spectroscopy studies of (La0.70−xNdx)Sr0.30Mn0.70Cr0.30O3 (x≤0.30) perovskite compounds

The transport properties of Nd-doped perovskite materials (La_0.7−xNd_x)Sr_0.3Mn_0.7Cr_0.3O₃ (x≤0.30) were investigated using impedance spectroscopy techniques over a wide range of temperatures and frequencies. AC conductance analyses indicate that the conduction mechanism is strongly dependent on temperature and frequency. The DC conductance plots can be described using the small polaron hopping (SPH) model, with an apparent reduction of the polaron activation energy below the Curie temperature T_C. Complex impedance plots exhibit semicircular arcs described by an electrical equivalent circuit. Off-centered semicircular impedance plots show that the Nd-doped compounds obey to a non-Debye relaxation process. The conductivity of grains and grain-boundaries has been estimated. The activation energies calculated from the conductance and from time relaxation analyses are comparable. This indicates that the same type of charge carriers is responsible for both the electrical conduction and relaxation phenomena.     

Sensitivity analysis to perturbations of the weight of a subset of items: The knapsack case study

In this paper, we study the sensitivity of the optimum to perturbations of the weight of a subset of items of both the knapsack problem (denoted KP) and knapsack sharing problem (denoted KSP). The sensitivity interval of the weight associated to an item is characterized by two limits, called lower and upper values, which guarantee the optimality of the solution at hand whenever the new weight’s value belongs to such an interval. For each perturbed weight, we try to establish approximate values of the sensitivity interval whenever the original problem is solved. We do it by applying a dynamic programming method where all established results require a negligible runtime. First, two cases are studied when considering an optimal solution of KP: (i) the case in which all perturbations are (non)negatives and (ii) the general case in which the set of the perturbed items is divided into two disjoint subsets (the first subset contains the nonnegative perturbations and the second one represents the subset of negative perturbations). Second, we show how we can adapt the results of KP to the KSP. All established results require a negligible runtime which grows the interest of such a study. Finally, for each of these problems, we will see the impact of the established results on an example while considering the various cases.     

Development and validation of a turbidimetry method for hydrogen sulphide evaluation in effluents of phosphoric acid production unit

The aim of this study was to develop and validate a simple, new and efficient method for the analysis of hydrogen sulphide (H₂S) released from the effluents of phosphoric acid production units. H₂S sampling was performed by absorption of the industrial gas into a cadmium acetate absorbing solution. The formed cadmium sulphide (CdS), as a result of a chemical reaction, was analysed by turbidimetry. A methodical validation study of the proposed method was performed according to the requirements of ISO 17025 standards. The proposed method was demonstrated to be precise, linear and accurate over a concentration range of 6.91–92.16 mg/l. Detection and quantification limits were equal to 5.09 mg/l and 6.91 mg/l, respectively. Sample analysis had to be performed within 48 h of the sampling step. The turbidimetry method was applied successfully to the industrial gaseous effluents and can be considered as an economical alternative to the iodometric method.     

Evaluating the photovoltaic properties of two conjugated polymers synthesized by Suzuki polycondensation and direct C–H activation

Two conjugated polymers HXS-1 and PDFCDTBT were prepared by direct C–H activation and Suzuki polycondensation and their chemical structures were characterized by 1H NMR spectroscopy.The molecular weight of conjugated polymer synthesized by direct C–H activation is lower than the corresponding polymers prepared by Suzuki polycondensation.Conjugated polymers synthesized by direct C–H activation have considerable solubility in common organic solvents and form amorphous film.The photovoltaic property of conjugated polymers synthesized by direct C–H activation is inferior to the corresponding polymers synthesized by Suzuki polycondensation.     

Concentration of fish serum albumin (FSA) in the aqueous extract of Indonesian Perciformes fishes’ muscle tissue

Fish serum albumin (FSA) is an aquatic resource that has potential to be developed as nutraceutical. Therefore, research was undertaken to assess albumin levels in the aqueous extract of muscle tissue of several Perciformes commonly available at a local fish market in Indonesia. Three random replicates for each of 17 Perciformes species were collected and assessed for their FSA content by application of a reversed-phase (C4) HPLC analytical method. Results of these analyses showed that the albumin concentration of the extracts was in the range 3.49–12.61 g/L, and that they varied significantly (P < 0.05) between species and families. This finding may mean that FSA levels are species and family dependent, something that could be investigated in future studies. As fishes from the family Scrombidae showed the highest concentration (12.61 g/L) of FSA, they would likely have the most value as a source for production of albumin-based nutritional and/or clinical products.