Dehydrated aqueous two-phase system micro-domains retain their shape upon rehydration to allow patterned reagent delivery to cells

Aqueous reagent solution micro-domains with sharp boundaries and defined shapes are created over cell monolayers within an immiscible bulk aqueous phase through rehydration of freestanding and portable dried reagent patches of the corresponding shape. This is in contrast to typical dissolution of reagent tablets or lyophilized biopolymer patches in aqueous solutions where no discernible reagent solution patterns are formed upon their full hydration. The key to enable the engineering of such stable reagent solution micro-domains is to formulate the reagent patches with polymers that form an aqueous two-phase system (ATPS) upon hydration by the bulk aqueous phase. This paper demonstrates this concept using dried reagent patches that incorporate dextran (DEX) and a bulk aqueous phase comprised of cell culture medium containing poly(ethylene) glycol (PEG). For reagents that prefer to partition in the DEX phase of the resulting ATPS, this procedure results in micro-patterned localization of reagent solution only to regions of the cell monolayer covered with the rehydrated DEX patch. The types of aqueous reagent solution micro-domain shapes that can be formed by the rehydration of such freestanding DEX-reagent patches are surprisingly broad and can be readily controlled by use of different templates for dehydrating the DEX solutions or even by cutting flat patches. The utility of the method is demonstrated through localized delivery of fluorescent molecules and enzymes for cell detachment. The patterned enzymatic detachment of cells enables convenient wound healing assays where cell monolayers can be wounded in different shapes dictated by the silhouette of the original DEX-reagent patchesclose1

RSC Adv.

Developing new biomaterials is an active research area owing to their applications in regenerative medicine, tissue engineering and drug delivery. Elastin-like polypeptides (ELPs) are good candidates for these applications because they are biosourced, biocompatible and biodegradable. With the aim of developing ELP-based micelles for drug delivery applications we have synthesized 15 acyl-ELP compounds by conjugating myristic, palmitic, stearic, oleic or linoleic acid to the N-terminus of three ELPs differing in molar mass. The ELP-fatty acid conjugates have interesting solution behavior. They form micelles at low temperatures and aggregate above the cloud point temperature (Tcp). The critical micelle concentration depends on the fatty acid nature while the micelle size is mainly determined by the ELP block length. We were able to show that ELPs were better hydrated in the micelles than in their individual state in solution. The micelles are stable in phosphate-buffer saline at temperatures below the Tcp, which can vary between 20 °C and 38 °C depending on the length or hydrophilicity of the ELP. Acyl-ELP micelles were loaded with the small hydrophobic molecule Nile red. The encapsulation efficiency and release kinetics showed that the best loading conditions were achieved with the largest ELP conjugated to stearic acid

Oxidative Transformation of Dihydroflavonols and Flavan-3-ols by Anthocyanidin Synthase from Vitis vinifera

Twelve polyphenols from three distinct families (dihydroflavonols, flavan-3-ols, and flavanones) were studied as potential substrates of anthocyanidin synthase from Vitis vinifera (VvANS). Only flavan-3-ols of (2R,3S) configuration having either a catechol or gallol group on ring B are accepted as substrates. Only dihydroflavonols of (2R,3R) configuration are accepted as substrates, but a catechol or gallol group is not mandatory. Flavanones are not substrates of VvANS. HPLC and MS/MS analyses of the enzymatic products showed that the VvANS-catalyzed oxidative transformation of (+)-dihydroflavonols, such as dihydroquercetin, dihydrokaempferol and dihydromyricetin, leads only to the corresponding flavonols. Among the flavan-3-ols recognized as substrates, (+)-gallocatechin was only transformed into delphinidin by VvANS, whereas (+)-catechin was transformed into three products, including two major products that were an ascorbate–cyanidin adduct and a dimer of oxidized catechin, and a minor product that was cyanidin. Data from real-time MS monitoring of the enzymatic transformation of (+)-catechin suggest that its products are all derived from the initial C3-hydroxylation intermediate, i.e., a 3,3-gem-diol, and their most likely formation mechanism is discussed

Gap Junctions Study Using a Microfluidic Sensor

Cells in tissues and organs coordinate their activities by communicating with each other, and this communication is mediated by specialized channels, called gap junctions. There are two types of gap junction communications both governed by potential gradients: molecular diffusion and electrolyte transport. Most cells are known to express multiple connexins that form different types of gap junctions. To date, there is no known gap junction blocker which specifically blocks one kind of gap junction channel. Therefore, we have developed a microfluidic sensor for studying intercellular communication in a high throughput manner. The concept of the microfluidic sensor is based on the ability for a multi-stream channel to partition a cell monolayer with different chemically loaded solutions. Numerical simulations were conducted to characterize the performance of the chip in terms of multiple flow control, interface stability and fluid exchange time. The flexibility of the sensor allowed the characterization of molecular diffusion and electrical coupling. The study of intercellular diffusion of fluorescent probes in Normal Rat Kidney cells confirmed the size dependent permeability through the Cx43. With the aid of a numerical algorithm, we were able to extract the diffusion rate of molecular probes from fluorescent microscopy recordings. The testing of different molecular size fluorescent dyes showed increasing diffusion rates with decreasing molecular weight. A similar experimental configuration gave us the ability to screen different gap junction blockers. The testing of showed homogeneous reduction of the diffusion rate for all tested dyes, while MFA displayed a charge dependent potency. The ability for the sensor to test different dyes or different blockers was also demonstrated in detail. We also measured the electrical intercellular communication via the same microfluidic chip using a sucrose gap configuration. The use of a non-conductive solution in the middle stream of the tri-stream channel allowed the constriction of electrolyte transport through gap junction channels. Both 1-Heptanol and 2-APB reversibly reduced gap junction communication. The gap junction blockers 1-Heptanol and 2-APB provided distinct kinetics of electrolyte transport alteration, suggesting the presence of two different blocking mechanisms. We also demonstrated the ability to screen candidate blockers such as the ion channel inhibitor, GsMTx-4. The application GsMTx-4 did not show an effect on the gap junction channels. Finally, we studied the effect of the cell substrate stiffness on the gap junctions' activity. The substrate was coated with a layer of Polydimethylsiloxane (PDMS), presenting different stiffness, prior to experimentation. The study of the molecular diffusion through cells cultured on substrates of different stiffness showed an increase of the molecular permeability with decreasing the substrate stiffness. To understand the link between intercellular diffusion of molecular probes and substrate stiffness, we immunostained proteins playing a role in substrate sensing, such as actin filaments and in the cell-cell junction such as ZO-1. The results showed an increased number of ZO-1 complexes for softer substrates and an increase of the Cx43 complexes at the cell-cell junction, which was responsible for facilitating gap junction mediated molecular diffusion

DEVICE AND METHOD FOR SINGLE CELL SCREENING BASED ON INTER-CELLULAR COMMUNICATION

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Etude des déterminants de la prescription des antivitamines K dans les structures de soins de longue durée du Finistère en prévention primaire d'accident vasculaire cérébral ischémique chez les sujets de plus de 75 ans en fibrillation auriculaire non valvulaire

BREST-BU Médecine-Odontologie (290192102) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Détection par spectrométrie de masse des O-glycans de la région charnière des IgA1 et relation avec leur capacité à déposer dans le mésangium rénal

International audienc