resonance energy transfer
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Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 199
Author(s):  
Lídia Ballell-Hosa ◽  
Elisabet González-Mira ◽  
Hector Santana ◽  
Judit Morla-Folch ◽  
Marc Moreno-Masip ◽  
...  

Topical delivery has received great attention due to its localized drug delivery, its patient compliance, and its low risk for side effects. Recent developments have focused on studying new drug delivery systems as a strategy for addressing the challenges of current topical treatments. Here we describe the advances on an innovative drug delivery platform called DELOS nanovesicles for topical drug delivery. Previously, the production of DELOS nanovesicles demonstrated potentiality for the topical treatment of complex wounds, achieving well-tolerated liquid dispersions by this route. Here, research efforts have been focused on designing these nanocarriers with the best skin tolerability to be applied even to damaged skin, and on exploring the feasibility of adapting the colloidal dispersions to a more suitable dosage form for topical application. Accordingly, these drug delivery systems have been efficiently evolved to a hydrogel using MethocelTM K4M, presenting proper stability and rheological properties. Further, the integrity of these nanocarriers when being gellified has been confirmed by cryo-transmission electron microscopy and by Förster resonance energy transfer analysis with fluorescent-labeled DELOS nanovesicles, which is a crucial characterization not widely reported in the literature. Additionally, in vitro experiments have shown that recombinant human Epidermal Growth Factor (rhEGF) protein integrated into gellified DELOS nanovesicles exhibits an enhanced bioactivity compared to the liquid form. Therefore, these studies suggest that such a drug delivery system is maintained unaltered when hydrogellified, becoming the DELOS nanovesicles-based hydrogels, an advanced formulation for topical use.


2022 ◽  
Author(s):  
Pengchao Wang ◽  
Guangming Zhang ◽  
Zeling Xu ◽  
Zhe Chen ◽  
Xiaohong Liu ◽  
...  

Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.


Sensors ◽  
2022 ◽  
Vol 22 (2) ◽  
pp. 561
Author(s):  
Andreia C. M. Rodrigues ◽  
Maria Vittoria Barbieri ◽  
Marco Chino ◽  
Giuseppe Manco ◽  
Ferdinando Febbraio

The development of faster, sensitive and real-time methods for detecting organophosphate (OP) pesticides is of utmost priority in the in situ monitoring of these widespread compounds. Research on enzyme-based biosensors is increasing, and a promising candidate as a bioreceptor is the thermostable enzyme esterase-2 from Alicyclobacillus acidocaldarius (EST2), with a lipase-like Ser–His–Asp catalytic triad with a high affinity for OPs. This study aimed to evaluate the applicability of Förster resonance energy transfer (FRET) as a sensitive and reliable method to quantify OPs at environmentally relevant concentrations. For this purpose, the previously developed IAEDANS-labelled EST2-S35C mutant was used, in which tryptophan and IAEDANS fluorophores are the donor and the acceptor, respectively. Fluorometric measurements showed linearity with increased EST2-S35C concentrations. No significant interference was observed in the FRET measurements due to changes in the pH of the medium or the addition of other organic components (glucose, ascorbic acid or yeast extract). Fluorescence quenching due to the presence of paraoxon was observed at concentrations as low as 2 nM, which are considered harmful for the ecosystem. These results pave the way for further experiments encompassing more complex matrices.


2022 ◽  
Vol 12 ◽  
Author(s):  
Ruslan Kalendar ◽  
Akmaral Baidyussen ◽  
Dauren Serikbay ◽  
Lyudmila Zotova ◽  
Gulmira Khassanova ◽  
...  

The proposed method is a modified and improved version of the existing “Allele-specific q-PCR” (ASQ) method for genotyping of single nucleotide polymorphism (SNP) based on fluorescence resonance energy transfer (FRET). This method is similar to frequently used techniques like Amplifluor and Kompetitive allele specific PCR (KASP), as well as others employing common universal probes (UPs) for SNP analyses. In the proposed ASQ method, the fluorophores and quencher are located in separate complementary oligonucleotides. The ASQ method is based on the simultaneous presence in PCR of the following two components: an allele-specific mixture (allele-specific and common primers) and a template-independent detector mixture that contains two or more (up to four) universal probes (UP-1 to 4) and a single universal quencher oligonucleotide (Uni-Q). The SNP site is positioned preferably at a penultimate base in each allele-specific primer, which increases the reaction specificity and allele discrimination. The proposed ASQ method is advanced in providing a very clear and effective measurement of the fluorescence emitted, with very low signal background-noise, and simple procedures convenient for customized modifications and adjustments. Importantly, this ASQ method is estimated as two- to ten-fold cheaper than Amplifluor and KASP, and much cheaper than all those methods that rely on dual-labeled probes without universal components, like TaqMan and Molecular Beacons. Results for SNP genotyping in the barley genes HvSAP16 and HvSAP8, in which stress-associated proteins are controlled, are presented as proven and validated examples. This method is suitable for bi-allelic uniplex reactions but it can potentially be used for 3- or 4-allelic variants or different SNPs in a multiplex format in a range of applications including medical, forensic, or others involving SNP genotyping.


2022 ◽  
Vol 12 ◽  
Author(s):  
Frédéric Larbret ◽  
Pierric Biber ◽  
Nicholas Dubois ◽  
Stoyan Ivanov ◽  
Laurence Lafanechere ◽  
...  

Actin networks are dynamically regulated through constant depolymerization and polymerization cycles. Although the fundamental mechanisms that govern these processes have been identified, the nature and role of post-translational modifications (PTMs) of actin and actin regulatory proteins are not completely understood. Here, we employed Actin CytoFRET, a method that we developed for real time detection of fluorescence resonance energy transfer (FRET) signals generated by actin dynamics, to screen a small library of PTM-interfering compounds on a biosensor leukemic T cell line. This strategy led to the identification of small molecule inhibitors of deubiquitinating enzymes (DUBs) as potent inducers of actin polymerization and blockers of chemotactic cell migration. The examination of the underlying mechanism further revealed that the actin depolymerizing protein cofilin represents a major effector of DUB inhibitor (DUBi)-induced actin reorganization. We found that DUB blockade results in the accumulation of polyubiquitinated proteins and ROS production, associated with cofilin oxidation and dephosphorylation on serine 3, which provokes uncontrolled actin polymerization impairing cell migration. Together, our study highlights DUBs as novel regulators of actin dynamics through ROS-dependent cofilin modulation, and shows that DUBi represent attractive novel tools to impede leukemic cell migration.


2022 ◽  
Author(s):  
Shiqi Liuye ◽  
Shiqiang Cui ◽  
Mengmeng Lu ◽  
Shouzhi Pu

Abstract Photo-controlled fluorescent switching is of great utility in fluorescence sensors, reversible data storage, and logic circuit, based on their modifiable emission intensity and spectra. In this work, a novel photo-controlled reversible fluorescent switching system was constructed based on photochromic diarylethene (DT) molecular modified fluorescent carbon dots (CDs). The fluorescent CDs acted as fluorescent donors and the photochromic diarylethene molecular functioned as acceptors in this fluorescent switching system. The fluorescence modulation efficiency of the fluorescent switching was determined to be 97.1%. The result was attributable to Förster resonance energy transfer (FRET) between the CDs and the diarylethene molecular. The fluorescent switching could undergo 20 cycles without significant decay.


2022 ◽  
Vol 15 (1) ◽  
pp. 73
Author(s):  
Sang-Hyun Ahn ◽  
Jung-Soo Suh ◽  
Yoon-Kwan Jang ◽  
Heon-Su Kim ◽  
Gyu-Ho Choi ◽  
...  

Rhynchosia volubilis, a small black bean, has been used as a traditional remedy to treat diseases and maintain health in East Asia, but its cellular effects and molecular mechanisms are not fully understood. The purpose of this study was to investigate the effect of ethanol extract from Rhynchosia volubilis (EERV) on cell survival and to elucidate the biochemical signaling pathways. Our results showed that EERV stimulated the cyclic AMP (cAMP) signal revealed by a fluorescent protein (FP)-based intensiometric sensor. Using a Förster resonance energy transfer (FRET)-based sensor, we further revealed that EERV could activate PKA and ERK signals, which are downstream effectors of cAMP. In addition, we reported that EERV could induce the phosphorylation of CREB, a key signal for cell survival. Thus, our results suggested that EERV protects against apoptosis by activating the cell survival pathway through the cAMP-PKA/ERK-CREB pathway.


2022 ◽  
Vol 905 ◽  
pp. 169-173
Author(s):  
Hui Ping Xi ◽  
Xiu Wei Fang ◽  
Bao Hua Wu

In Britton Robinson (B-R) buffer solution with pH = 7.00 and sodium dodecyl sulfate (SDS) medium, an effective energy transfer between acridine orange (AO) and Rhodamine B (RB) can occur, which can enhance the fluorescence of RB. The addition of Gemifloxacin (GMFX) can quench the fluorescence of RB. So a new method for the indirect determination of Gemifloxacin was established by AO-RB fluorescence resonance energy transfer. This method was applied to the determination of Gemifloxacin tablets. The results were consistent with those of high performance liquid chromatography (HPLC) . Experiments show that this method is simple, rapid, accurate and sensitive.


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