scholarly journals A FRET Approach to Detect Paraoxon among Organophosphate Pesticides Using a Fluorescent Biosensor

Sensors ◽  
2022 ◽  
Vol 22 (2) ◽  
pp. 561
Author(s):  
Andreia C. M. Rodrigues ◽  
Maria Vittoria Barbieri ◽  
Marco Chino ◽  
Giuseppe Manco ◽  
Ferdinando Febbraio
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Triad ◽  
Thermostable Enzyme ◽  
In Situ Monitoring ◽  
Promising Candidate ◽  
Fluorescent Biosensor ◽  
Alicyclobacillus Acidocaldarius ◽  
Significant Interference

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.

scholarly journals TCR–pMHC bond conformation controls TCR ligand discrimination

2019 ◽  
Vol 17 (3) ◽  
pp. 203-217 ◽  
Author(s):  
Dibyendu K. Sasmal ◽  
Wei Feng ◽  
Sobhan Roy ◽  
Peter Leung ◽  
Yanran He ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Feedback Loop ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Intrinsic Property ◽  
Unanswered Question ◽  
Structural Differences ◽  
Self Peptides

Abstract A major unanswered question is how a TCR discriminates between foreign and self-peptides presented on the APC surface. Here, we used in situ fluorescence resonance energy transfer (FRET) to measure the distances of single TCR–pMHC bonds and the conformations of individual TCR–CD3ζ receptors at the membranes of live primary T cells. We found that a TCR discriminates between closely related peptides by forming single TCR–pMHC bonds with different conformations, and the most potent pMHC forms the shortest bond. The bond conformation is an intrinsic property that is independent of the binding affinity and kinetics, TCR microcluster formation, and CD4 binding. The bond conformation dictates the degree of CD3ζ dissociation from the inner leaflet of the plasma membrane via a positive calcium signaling feedback loop to precisely control the accessibility of CD3ζ ITAMs for phosphorylation. Our data revealed the mechanism by which a TCR deciphers the structural differences among peptides via the TCR–pMHC bond conformation.

In situ manipulation of fluorescence resonance energy transfer between quantum dots and monolayer graphene oxide by laser irradiation

Nanoscale ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 1236-1244 ◽  
Author(s):  
Wenjun He ◽  
Chengbing Qin ◽  
Zhixing Qiao ◽  
Yani Gong ◽  
Xiaorong Zhang ◽  
...  
Keyword(s):  
Quantum Dots ◽  
Graphene Oxide ◽  
Energy Transfer ◽  
Laser Irradiation ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Monolayer Graphene ◽  
Fluorescence Resonance

Fluorescence resonance energy transfer between CdSeTe/ZnS quantum dots and monolayer graphene oxide is in situ manipulated by laser irradiation.

scholarly journals Endogenous microRNA Triggered Enzyme-free DNA Logic Self-assembly for Amplified Bioimaging and Enhanced Gene Therapy via in Situ Generation of siRNAs

2021 ◽  
Author(s):  
Qinghua Jiang ◽  
Shuzhen Yue ◽  
Kaixin Yu ◽  
Tian Tian ◽  
Jian Zhang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Self Assembly ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Amplification ◽  
High Sensitivity ◽  
Free Dna ◽  
Therapy Effect ◽  
In Situ Generation

Abstract BackgroundSmall interfering RNA (siRNA) has emerged as a kind of promising therapeutic agents for cancer therapy. However, the off-target effect and degradation are the main challenges for siRNAs delivery. Herein, an enzyme-free DNA amplification strategy initiated by a specific endogenous microRNA has been developed for in situ generation of siRNAs with enhanced gene therapy effect on cervical carcinoma.MethodsThis strategy contains three DNA hairpins (H1, H2/PS and H3) which can be triggered by microRNA-21 (miR-21) for self-assembly of DNA nanowheels (DNWs). Notably, this system is consistent with the operation of a DNA logic circuitry containing cascaded “AND” gates with feedback mechanism. Accordingly, a versatile biosensing and bioimaging platform is fabricated for sensitive and specific analysis of miR-21 in HeLa cells via fluorescence resonance energy transfer (FRET). Meanwhile, since the vascular endothelial growth factor (VEGF) antisense and sense sequences are encoded in hairpin reactants, the performance of this DNA circuit leads to in situ assembly of VEGF siRNAs in DNWs, which can be specifically recognized and cleaved by Dicer for gene therapy of cervical carcinoma. ResultsThe proposed isothermal amplification approach exhibits high sensitivity for miR-21 with a detection limit of 0.25 pM and indicates excellent specificity to discriminate target miR-21 from the single-base mismatched sequence. Furthermore, this strategy achieves accurate and sensitive imaging analysis of the expression and distribution of miR-21 in different living cells. To note, compared to naked siRNAs alone, in situ siRNA generation shows a significantly enhanced gene silencing and anti-tumor effect due to the high reaction efficiency of DNA circuit and improved delivery stability of siRNAs.ConclusionThe endogenous miRNA-activated DNA circuit provides an exciting opportunity to construct a general nanoplatform for precise cancer diagnosis and efficient gene therapy, which has an important significance in clinical translation.

scholarly journals Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease

2016 ◽  
Vol 311 (3) ◽  
pp. G571-G580 ◽  
Author(s):  
Guoxian Wei ◽  
Na Tian ◽  
Roland Siezen ◽  
Detlef Schuppan ◽  
Eva J. Helmerhorst
Keyword(s):  
Celiac Disease ◽  
Serine Proteases ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Triad ◽  
Local Alignment ◽  
Peptide Substrates ◽  
Food Grade ◽  
Search Tool ◽  
Rothia Mucilaginosa

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

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