Search Results (306)

von Willebrand factor (vWF) is a large glycoprotein in the circulation system, which senses hydrodynamic force at vascular injuries and then recruits platelets in assembling clots. How vWF mechanosenses shear flow for molecular unfolding is an important topic. Here, a Förster resonance energy transfer (FRET) biosensor was developed to monitor vWF conformation change to hydrodynamic force. The vWF-based biosensor is anchored on the cell surface, in which the A2 domain is flanked with a FRET pair. With 293T cells seeded into microfluidic channels, 2.8 dyn/cm² of shear force (i.e., 28 μN/cm², or 264.1/s in shear rate) induced a remarkable FRET change (~60%) in 30 min. A gradient micro-shear below 2.8 dyn/cm² demonstrated FRET responses positively related to flow magnitudes, with 0.14 dyn/cm² (1.4 μN/cm²) inducing an obvious change (~16%). The FRET increases indicate closer positioning of A2’s two terminals in vWF or the addition of a more parallel orientation of the FRET pair, supported with the high FRET of the A2-only-based biosensor, which probably resulted from flow-induced A2 dissociation from vWF intramolecular binding such as that in A1/A3 domains. Interestingly, gradient flow increases from 2.8 to 28 dyn/cm² led to decreasing FRET changes, suggesting the second-level unfolding in the A2 domain. The LOCK-vWF biosensor with bridged A2 two terminals or an A2-only biosensor could not sense the shear, indicating a structure-flexible A2 and large vWF molecules that are important in the mechanosensation. In conclusion, the developed vWF-based biosensor demonstrated the high mechanosensation of vWF with two-level unfolding to shear force: the dissociation of the A2 domain from vWF intramolecular binding under a micro-shear, and then the unfolding of A2 in vWF under a higher shear; the FRET response to shear force at a very low scale may support the observed clot formation at microvascular wounds. This study provides new insights into the vWF’s mechanosensitive feature for its physiological functions and implicated disorders. Full article

Reconstituted high-density lipoprotein nanoparticles (NPs), which mimic the structure and function of endogenous human plasma HDL, hold promise as a robust drug delivery system. These nanoparticles, when loaded with appropriate agents, serve as powerful tools for targeted drug delivery. The fundamental challenge lies in controlling and estimating the actual drug load and the efficiency of drug release at the target. In this report, we present a novel approach based on enhanced Förster Resonance Energy Transfer (FRET) to assess particle load and monitor payload release. The NPs are labeled with donor molecules embedded in the lipid phase, while the spherical core volume is filled with acceptor molecules. Highly enhanced FRET efficiency to multiple acceptors in the NP core has been observed at distances significantly larger than the characteristic Förster distance (R₀). To confirm that the observed changes in donor and acceptor emissions are a result of FRET, we developed a theoretical model for nonradiative energy transfer from a single donor to multiple acceptors enclosed in a spherical core volume. The load-dependent shortening of the fluorescence lifetime of the donor correlated with the presence of a negative component in the intensity decay of the acceptor clearly demonstrates that FRET can occur at a large distance comparable to the nanoparticle size (over 100 Å). Comparison of theoretical simulations with the measured intensity decays of the donor and acceptor fluorophores constitute a new method for evaluating particle load. The observed FRET efficiency depends on the number of acceptors in the core, providing a simple way to estimate the nanoparticle load efficiency. Particle disintegration and load release result in a distinct change in donor and acceptor emissions. This approach constitutes a novel strategy for assessing NP core load, monitoring NP integrity, and evaluating payload release efficiency to target cells. Significants: In the last decade, nanoparticles have emerged as a promising strategy for targeted drug delivery, with applications ranging from cancer therapy to ocular neurodegenerative disease treatments. Despite their potential, a significant issue has been the real-time monitoring of these drug delivery vehicles within biological systems. Effective strategies for monitoring NP payload loading, NP integrity, and payload release are needed to assess the quality of new drug delivery systems. In our study, we have found that FRET-enabled NPs function as an improved method for monitoring these aspects currently missing from current drug delivery efforts. Full article

We established a real-time Förster resonance energy transfer (FRET) based assay to evaluate targeted drug delivery using polymeric micelles. Red fluorescent protein (RFP)-expressing E. coli cells were used as a test system to monitor the delivery of drug-fluorophore such as curcumin and umbelliferones (MUmb and AMC) encapsulated in the polymeric micellar formulations. The efficiency of the drug delivery was quantified using the FRET efficiency, measured as the degree of energy transfer from the drug to the RFP. FRET efficiency directly provides the determination of the delivery efficacy, offering a versatile platform adaptable to various drugs and cell types. We used polymer micelles as a carrier for targeted delivery of fluorescent drugs to bacterial cells expressing RFP. The physicochemical characterization of the interaction between the drugs and the micelles including spectral properties, and the solubility and binding constants, were determined. We revealed a stronger affinity of MUmb for heparin-based micelles (K_d~10⁻⁵ M) compared to chitosan-based micelles (K_d~10⁻⁴ M), underscoring the influence of polymer composition on drug loading efficiency. For micelles containing MUmb, a FRET efficiency significantly exceeds (by three times) the efficiency for non-micellar MUmb, which have minimal penetration into bacterial cells. The most noticeable effect was observed with the use of the micellar curcumin providing pronounced activation of the RPF fluorescence signal, due to the interaction with curcumins (fluorophore-donor). Curcumin delivery using Chit5-OA micelle resulted in a 115% increase in RFP fluorescence intensity, and Hep-LA showed a significant seven-fold increase. These results highlight the significant effect of micellar composition on the effectiveness of drug delivery. In addition, we have developed a visual platform designed to evaluate the effectiveness of a pharmaceutical product through the visualization of the fluorescence of a bacterial culture on a Petri dish. This method allows us to quickly and accurately assess the penetration of a drug into bacteria, or those located inside other cells, such as macrophages, where the intercellular latent forms of the infection are located. Micellar formulations show enhanced antibacterial activity compared to free drugs, and formulations with Hep-OA micelles demonstrate the most significant reduction in E. coli viability. Synergistic effects were observed when combining curcumin and MUmb with moxifloxacin, resulting in a remarkable 40–50% increase in efficacy. The presented approach, based on the FRET test system with RFP expressed in the bacterial cells, establishes a powerful platform for development and optimizing targeted drug delivery systems. Full article

Nitroaromatic-explosives (NEs) not only threaten global security but are also recognized as a highly toxic pollutant. Metal–organic framework Zn-M_s (Zn-M₁, Zn-M₂) were synthesized in this study via the coordination-driven self-assembly of Zn ions and a carbazole-based ligand L containing an aldehyde group. They inherited the excellent fluorescence performance of ligand L and could work as a fluorescent sensor for detecting picric acid (PA) at low concentrations. Zn-M_s showed an emission at 450 nm and exhibited a higher fluorescence quenching efficiency toward PA than other related NEs. The results suggest that the fluorescent response might be attributed to the inner filter effect (IFE); Förster resonance energy transfer (FRET); and possibly, photo-induced electron transfer (PET). In addition, the critical role of the aldehyde group as a recognition site was corroborated using a post-modification strategy. Full article

The binding interactions between okadaic acid (OA) aptamers and OA molecules are crucial for developing effective detection methods. This study aims to identify the recognition site and establish a reliable detection protocol through computational simulations and experimental validations. After determining the target sequence (OA-2), molecular docking simulations using Sybyl-X and H-dock were conducted to predict the binding affinity and interaction sites of OA aptamers with their targets. These predictions were subsequently validated through experiments based on the Förster resonance energy transfer (FRET) principle. The combined approach not only confirmed the computational predictions, identifying the “major region” as the recognition basis of OA-2, but also provided deeper insights into the binding mechanisms. Subsequently, a classical AuNPs-aptamer colorimetric detection method was established based on the OA-2 sequence and applied to the detection of real shellfish samples, achieving a limit of quantification (LOQ) of 5.0 μg kg⁻¹. The recoveries of OA in spiked samples ranged from 79.0% to 122.9%, with a relative standard deviation (RSD) of less than 14.7%. The results of this study contribute to the development of robust detection methods for OA aptamer–target interactions, enhancing the potential for practical applications in toxin detection and monitoring. Full article

Three neutral iridium complexes Ir1–Ir3 were synthesized using diphenylphosphoryl-substituted 2-phenylpyridine derivatives as the cyclometalating ligand and picolinic acid as the auxiliary ligand. They exhibited significant aggregation-induced phosphorescent emission (AIPE) properties in H₂O/THF and were successfully used as bi-responsive luminescent sensors for the detection of picric acid (PA) and Fe³⁺ in aqueous media. Ir1–Ir3 possesses high efficiency and high selectivity for detecting PA and Fe³⁺, with the lowest limit of detection at 59 nM for PA and 390 nM for Fe³⁺. Additionally, the complexes can achieve naked-eye detection of Fe³⁺ in aqueous media. Ir1–Ir3 exhibit excellent potential for practical applications in complicated environments. The detection mechanism for PA is attributed to photo-induced electron transfer (PET) and Förster resonance energy transfer (FRET), and the detection mechanism for Fe³⁺ may be explained by PET and the strong interactions between Fe³⁺ and the complexes. Full article

Advancements in cellular imaging have significantly enhanced our understanding of membrane potential and Ca²⁺ dynamics, which are crucial for various cellular processes. Voltage-sensitive dyes (VSDs) are pivotal in this field, enabling non-invasive, high-resolution visualization of electrical activity in cells. This review discusses the various types of VSDs, including electrochromic, Förster Resonance Energy Transfer (FRET)-based, and Photoinduced Electron Transfer (PeT)-based dyes. VSDs are essential tools for studying mitochondrial activity and neuronal function and are frequently used in conjunction with Ca²⁺ indicators to elucidate the complex relationship between membrane potential and Ca²⁺ fluxes. The development of novel dyes with improved photostability and reduced toxicity continues to expand the potential of VSDs in biomedical research. This review underscores the importance of VSDs in advancing our understanding of cellular bioenergetics, signaling, and disease mechanisms. Full article

Laminopathies represent a wide range of genetic disorders caused by mutations in gene-encoding proteins of the nuclear lamina. Altered nuclear mechanics have been associated with laminopathies, given the key role of nuclear lamins as mechanosensitive proteins involved in the mechanotransduction process. To shed light on the nuclear partners cooperating with altered lamins, we focused on Src tyrosine kinase, known to phosphorylate proteins of the nuclear lamina. Here, we demonstrated a tight relationship between lamin A/C and Src in skin fibroblasts from two laminopathic patients, assessed by advanced imaging-based microscopy techniques. With confocal laser scanning and Stimulated Emission Depletion (STED) microscopy, a statistically significant higher co-distribution between the two proteins was observed in patients’ fibroblasts. Furthermore, the time-domain fluorescence lifetime imaging microscopy, combined with Förster resonance energy transfer detection, demonstrated a decreased lifetime value of Src (as donor fluorophore) in the presence of lamin A/C (as acceptor dye) in double-stained fibroblast nuclei in both healthy cells and patients’ cells, thereby indicating a molecular interaction that resulted significantly higher in laminopathic cells. All these results demonstrate a molecular interaction between Src and lamin A/C in healthy fibroblasts and their aberrant interaction in laminopathic nuclei, thus creating the possibilities of new diagnostic and therapeutic approaches for patients. Full article

DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair. We identified HROB independently as a nuclear protein whose expression is co-regulated with various DNA replication factors. Accordingly, the HROB protein level showed a maximum in S phase and a downregulation in quiescence. Structural prediction and homology searches revealed that HROB is a largely intrinsically disordered protein bearing a helix-rich region and a canonical oligonucleotide/oligosaccharide-binding-fold motif that originated early in eukaryotic evolution. Employing a flow cytometry Förster resonance energy transfer (FRET) assay, we detected associations between HROB and proteins of the DNA replication machinery. Moreover, ectopic expression of HROB protein led to an almost complete shutdown of DNA replication. The available data imply a function for HROB during DNA replication across barriers such as ICLs. Full article

Accurate and selective monitoring of thiamine levels in multivitamin supplements is essential for preventing deficiencies and ensuring product quality. To achieve this, a Förster resonance energy transfer (FRET) system using carbon dots (CDs) as energy donors and citrate-stabilized silver nanoparticles (AgNPs) as energy acceptors was developed. The aqueous synthesis of AgNPs using microwave irradiation was optimized to obtain efficient plasmonic nanoparticles for FRET applications, targeting maximal absorbance intensity, stability, and wavelength alignment. Using a central composite orthogonal design (CCOD), the optimal conditions were identified as a 12.5 min microwave reaction time, a Ag molar ratio of 0.72, and a pH of 8.28. The FRET sensing scheme was applied for thiamine determination, where the vitamin’s presence impaired the FRET process, restoring CDs’ photoluminescence (PL) emission in a concentration-dependent manner. To mitigate interference from other vitamins, PL kinetic data and excitation–emission matrix (EEM) data were analyzed using unfolded partial least-squares (U-PLS) with the subsequent application of the residual bilinearization technique (RBL), achieving high sensitivity and specificity for thiamine detection. This method demonstrated its accuracy and robustness by attaining a determination coefficient (R²) of 0.952 and a relative error of prediction (REP%) of 11%. This novel method offers highly sensitive and interference-free thiamine detection, with significant potential for a wide range of analytical applications. Full article

A Black Hole Quencher-3 (BHQ-3) derivative was synthesized through an azo-coupling reaction between Methylene Violet 3RAX and a tertiary aniline functionalized with a pendant primary amine, allowing subsequent peptide conjugation. The synthesized compounds were characterized using NMR, UV–vis absorption, fluorescence spectroscopy, and mass spectrometry. The spectral properties of a Cy5/BHQ-3 amine pair were investigated through titration experiments in PBS (pH 7.4). The results confirmed Förster Resonance Energy Transfer (FRET), along with additional dynamic quenching, as evidenced by the Stern–Volmer analysis. The Stern–Volmer constant (K_SV) was determined to be 1.40 × 10⁵ M⁻¹. These findings confirm the potential of this system for use in molecular probes and bioimaging applications. Full article

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamD^Y184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamD^R94, BamD^R97, and BamA^E127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex. Full article

The natural polyphenol resveratrol is a biologically active compound that interacts with DNA and affects the activity of some nuclear enzymes. Its effect on the interaction between nucleosomes and poly(ADP-ribose) polymerase-1 (PARP1) and on the catalytic activity of PARP1 was studied using Western blotting, spectrophotometry, electrophoretic mobility shift assay, and single particle Förster resonance energy transfer microscopy. Resveratrol inhibited PARP1 activity at micro- and sub-micromolar concentrations, but the inhibitory effect decreased at higher concentrations due to the aggregation of the polyphenol. The inhibition of PARP1 by resveratrol was accompanied by its binding to the enzyme catalytic center and a subsequent decrease in PARP1 affinity to nucleosomal DNA. Concurrent binding of talazoparib to the substrate binding pocket of PARP1, which occurs in the presence of resveratrol, restores the interaction of PARP1 with nucleosomes, suggesting that the binding sites of resveratrol and talazoparib overlap. The data suggest that resveratrol can be classified as a natural inhibitor of PARP1. Full article

Interactions between gold metallic nanoparticles and molecular dyes have been well described by the nanometal surface energy transfer (NSET) mechanism. However, the expansion and testing of this model for nanoparticles of different metal composition is needed to develop a greater variety of nanosensors for medical and commercial applications. In this study, the NSET formula was slightly modified in the size-dependent dampening constant and skin depth terms to allow for modeling of different metals as well as testing the quenching effects created by variously sized gold, silver, copper, and platinum nanoparticles. Overall, the metal nanoparticles followed more closely the NSET prediction than for Förster resonance energy transfer, though scattering effects began to occur at 20 nm in the nanoparticle diameter. To further improve the NSET theoretical equation, an attempt was made to set a best-fit line of the NSET theoretical equation curve onto the Au and Ag data points. An exhaustive grid search optimizer was applied in the ranges for two variables, $0.1\le C\le 2.0$ and $0\le \alpha \le 4$, representing the metal dampening constant and the orientation of donor to the metal surface, respectively. Three different grid searches, starting from coarse (entire range) to finer (narrower range), resulted in more than one million total calculations with values $C=2.0$ and $\alpha =0.0736$. The results improved the calculation, but further analysis needed to be conducted in order to find any additional missing physics. With that motivation, two artificial intelligence/machine learning (AI/ML) algorithms, multilayer perception and least absolute shrinkage and selection operator regression, gave a correlation coefficient, $R^2$, greater than $0.97$, indicating that the small dataset was not overfitting and was method-independent. This analysis indicates that an investigation is warranted to focus on deeper physics informed machine learning for the NSET equations. Full article

Background/Objectives: The study of oxidative stress in cells and ways to prevent it attract increasing attention. Antioxidant defense of cells can be activated by releasing the transcription factor Nrf2 from a complex with Keap1, its inhibitor protein. The aim of the work was to study the effect of the modular nanotransporter (MNT) carrying an R1 anti-Keap1 monobody (MNT_R1) on cell homeostasis. Methods: The murine hepatocyte AML12 cells were used for the study. The interaction of fluorescently labeled MNT_R1 with Keap1 fused to hrGFP was studied using the Fluorescence-Lifetime Imaging Microscopy–Förster Resonance Energy Transfer (FLIM-FRET) technique on living AML12 cells transfected with the Keap1-hrGFP gene. The release of Nrf2 from the complex with Keap1 and its levels in the cytoplasm and nuclei of the AML12 cells were examined using a cellular thermal shift assay (CETSA) and confocal laser scanning microscopy, respectively. The effect of MNT on the formation of reactive oxygen species was studied by flow cytometry using 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate. Results: MNT_R1 is able to interact with Keap1 in the cytoplasm, leading to the release of Nrf2 from the complex with Keap1 and a rapid rise in Nrf2 levels both in the cytoplasm and nuclei, ultimately causing protection of cells from the action of hydrogen peroxide. The possibility of cleavage of the monobody in endosomes leads to an increase in the observed effects. Conclusions: These findings open up a new approach to specifically modulating the interaction of intracellular proteins, as demonstrated by the example of the Keap1-Nrf2 system. Full article