Search Results (55)

Rare earth elements (REEs), located in the IIIB group of the periodic table, can be detected in very small quantities by sensitive detection techniques. REE labeling technologies utilize fluorescent labeling, magnetic labeling, atomic fluorescence labeling, fluorescence resonance energy transfer (FRET) labeling and radiolabeling. Widely used immunoassays related to REE-labeled technologies include time-resolved fluorescence immunofluorescence assay (TRFIA), inductively coupled plasma–mass spectrometry (ICP–MS)-based immunoassays, mass spectrometry flow-through (CyTOF), and upconversion nanoparticles (UCNPs). REE-labeled immunoassays have been widely used in various fields, such as biological analysis, biomarker detection and analysis of food detection techniques, as these assays can use low quantities of biological tissue, exhibit stability, can label materials, lack radioactivity and show multidetection capability. To provide researchers with a deeper understanding of the immunoassay technique used to label rare earth elements, this paper reviews its labeling principle, detection technology, and application. Full article

RhoB is an atypical Rho GTPase whose function is tightly linked to its subcellular localization and membrane trafficking, reflecting its unique post-translational modifications and association with endosomal membranes in addition to the plasma membrane. Despite its implication in membrane trafficking and cytoskeletal regulation, tools to directly monitor RhoB activity in space and time have been lacking. Here, we describe the development and validation of a single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensor that enables direct visualization of RhoB activity in living cells while preserving its native membrane-targeting determinants. The biosensor exhibits a large dynamic range and resolves spatially heterogeneous RhoB activity during leading-edge protrusion–retraction cycles in migrating mouse embryonic fibroblasts. To demonstrate the utility of this tool, we performed multiplex live-cell imaging with a previously developed near-infrared FRET biosensor for the exocytic Rho GTPase TC10. Quantitative morphodynamic and cross-correlation analyses reveal coordinated yet antagonistic spatiotemporal patterns of RhoB and TC10 activities at the leading edge and show that perturbation of TC10 regulation reorganizes their spatial coupling. Together, this work introduces a robust biosensor for RhoB and establishes a multiplex imaging framework to study the coordination of trafficking and signaling during cell migration. Full article

Fibroblast activation protein (FAP), a transmembrane serine protease expressed primarily in pathological conditions, plays a pivotal role in tumor progression. Despite extensive studies on FAP in solid tumors, its role in hematologic cancers, particularly lymphoid malignancies, remains underexplored. This study aimed to investigate the level and activity of soluble FAP (sFAP) in pre-therapeutic serum samples from 120 lymphoma patients. We measured sFAP serum levels using time-resolved immunofluorometric assay and sFAP activity with Förster resonance energy transfer assay. In addition, immunohistochemistry was used to analyze intratumoral FAP expression in tissue biopsies from a subset of B-cell lymphoma patients (n = 34). Notably, the results revealed significantly reduced circulating sFAP levels (p = 0.002) and activity (p < 0.001) in aggressive disease subtypes compared with indolent subtypes and healthy individuals. At the time of diagnosis, low sFAP activity correlated with inferior overall survival (both p < 0.001) in patients with the aggressive entities, suggesting altered FAP functionality in these tumors. Interestingly, measuring intratumoral FAP levels revealed an inverse pattern, with diffuse large B-cell lymphoma showing higher tissue FAP localization compared with follicular lymphoma (p < 0.001). These findings provide new insights into the biological and clinical significance of FAP in lymphoid malignancies, particularly highlighting the importance of sFAP activity as a potential prognostic marker in aggressive lymphoid malignancies. Full article

Protein–protein interactions (PPIs) play a crucial role in various biological processes, including signal transduction, transcriptional regulation, and metabolic pathways. Over the years, many methods have been developed to study PPIs, such as yeast two-hybrid (Y2H), co-immunoprecipitation (Co-IP), pull-down assays, and surface plasmon resonance (SPR). However, each of these techniques has its own limitations, including false positives, a lack of specific binding partners, and restricted interaction zones. Fluorescence resonance energy transfer (FRET) has emerged as a powerful technique for investigating PPIs, offering several advantages over traditional methods. Recent advancements in fluorescence microscopy have further enhanced its application in PPI studies. In this review, we summarize recent developments in FRET-based approaches and their applications in PPIs research over the past five years, including conventional FRET, time-resolved FRET (TR-FRET), fluorescence lifetime imaging microscopy-FRET (FLIM-FRET), single-molecule FRET (smFRET), fluorescence cross-correlation spectroscopy FRET (FCCS-FRET), and provide guidance on selecting the most appropriate method for PPIs studies. Full article

The study’s aim was to evaluate electroretinographic (ERG) alterations in Fragile X syndrome (FXS), FMR1 premutation carriers, and controls, and to explore correlations with peripheral blood FMRP expression levels and behavioral outcomes. ERG recordings were obtained using a handheld device across three stimulus protocols in 43 premutation carriers, 39 individuals with FXS, and 23 controls. Peripheral blood FMRP expression levels were quantified using TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer). Correlations were assessed with cognitive and behavioral measures including IQ (Intelligence Quotient), ABC_FX (Aberrant Behavior Checklist for Fragile X Syndrome), SNAP-IV (Swanson, Nolan, and Pelham Teacher and Parent Rating Scale), SEQ (Sensory Experiences Questionnaire), ADAMS (Anxiety, Depression, and Mood Scale), and the Vineland III Adaptive Behavior Scale standard score. Significant group differences were observed in multiple ERG parameters, particularly in 2 Hz b-wave amplitude (p = 0.0081), 2 Hz b-wave time to peak (p = 0.0164), 28.3 Hz flash combined amplitude (p = 0.0139), 3.4 Hz red/blue flash b-wave amplitude (p = 0.0026), and PhNR amplitude (p = 0.0026), indicating both outer and inner retinal dysfunction in FXS and premutation groups. Despite high test–retest reliability for ERG (ICC range = 0.71–0.92) and FMRP (ICC = 0.70), no correlation was found between ERG metrics and FMRP or behavioral measures. However, FMRP levels strongly correlated with IQ (ρ = 0.69, p < 0.0001) and inversely with behavioral impairment [ABC_FX (ρ = −0.47, p = 0.0041), SNAP-IV (ρ = −0.48, p = 0.0039), SEQ (ρ = −0.43, p = 0.0146), and the Vineland III standard score (ρ = 0.56, p = 0.0019)]. ERG reveals distinct retinal functional abnormalities in FMR1-related conditions but does not correlate with peripheral FMRP expression levels, highlighting the need for multimodal biomarkers integrating radiological, physiological, behavioral, and molecular measures. Full article

Time-resolved fluorescence techniques, such as fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation spectroscopy (FCS), and time-resolved fluorescence spectroscopy, are ideally suited for investigating molecular dynamics and interactions in biological and chemical systems. However, the analysis and interpretation of these datasets require advanced computational tools capable of handling diverse models and datasets. This paper presents a comprehensive software solution designed for model generation and analysis of time-resolved fluorescence data with a strong focus on fluorescence for quantitative structural analysis and biophysics. The software supports the integration of multiple fluorescence techniques and provides users with robust tools for performing complex model analysis across diverse experimental data. By enabling global analysis, model generation, data visualization, and sampling over model parameters, the software enhances the interpretability of intricate fluorescence phenomena. By providing flexible modeling capabilities, this solution offers a versatile platform for researchers to extract meaningful insights from time-resolved fluorescence data, aiding in the understanding of dynamic biomolecular processes. Full article

The receptor-binding domain (RBD) of SARS-CoV-2 spike protein is responsible for the recognition of the Angiotensin-Converting Enzyme 2 (ACE2) receptor in human cells and, thus, plays a critical role in viral infection. The therapeutic value of targeting this interaction has been proven by a sizable body of research investigating antibodies, small proteins, aptamers, and peptides. This study presents a novel peptide that impinges the interaction between RBD and ACE2. Starting from a very large pool of structurally designed peptides extracted from our database, PepI-Covid19, a diverse set of peptides were studied using molecular dynamics simulations. Ten of the most promising were chemically synthesized and validated both in vitro and in a cell-based assay. Our results indicate that one of the peptides (PEP10) exhibited the highest disruption of the RBD/ACE2 complex, effectively blocking the binding of two molecules and consequently inhibiting the SARS-CoV-2 spike-mediated cell entry of viruses pseudotyped with the spike of the D614G, Delta, and Omicron variants. PEP10 can potentially serve as a scaffold that can be further optimized for improved affinity and efficacy. Full article

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an “APPROACH” strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙μL⁻¹, 0.77 ng∙μL⁻¹, and 1.1 ng∙μL⁻¹, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙μL⁻¹ in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy. Full article

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study. Full article

To enhance the specificity and sensitivity, cut the cost, and realize joint detection of multiple indicators, an immunoassay system based on the technology of time-resolved fluorescence resonance energy transfer (TR-FRET) was studied. Due to the FRET of the reagent, the donor probe and acceptor probe emitted specific fluorescence to enhance specificity. Long-lifetime specific fluorescence from the acceptor probe was combined with time-resolved technology to enhance sensitivity. A xenon flash lamp and a photomultiplier tube (PMT) were selected as the light source and detector, respectively. A filter-switching mechanism was placed in the light path, so the fluorescence signal from the donor and acceptor was measured alternately. The instrument’s design is given, and some specificI parts are described in detail. Key technical specifications of the instrument and procalcitonin (PCT), C-reactive protein (CRP), and interleukin-6(IL-6) were tested, and the test results were presented subsequently. The CV value of the self-designed counting module is better than 0.01%, and the instrument noises for 620 nm and 665 nm are 41.44 and 10.59, respectively. When set at 37 °C, the temperature bias (B) is 0.06 °C, and the temperature fluctuation is 0.10 °C. The CV and bias are between ±3% and 5%, respectively, when pipetting volumes are between 10 μL and 100 μL. Within the concentration range of 0.01 nM to 10 nM, the luminescence values exhibit linear regression correlation coefficients greater than 0.999. For PCT detection, when the concentration ranges from 0.02 ng/mL to 50 ng/mL, the correlation coefficient of linear fitting exceeds 0.999, and the limit of quantification is 0.096 ng/mL. For CRP and IL-6, the detection concentration ranges from 0 ng/mL to 500 ng/mL and 0 ng/mL to 20 ng/mL, respectively, with limits of quantification of 2.70 ng/mL and 2.82 ng/mL, respectively. The experimental results confirm the feasibility of the technical and instrumental solutions. Full article

Edible berries such as the fruits of black chokeberry (Aronia melanocarpa (Michx.) Elliott) and bilberry (Vaccinium myrtillus L.) are considered to be rich in phenolic compounds, which are nowadays attracting great interest due to their promising health benefits. The main objective of our study was to investigate, for the first time, their inhibitory properties on Src tyrosine kinase activity, as this enzyme plays an important role in multiple cellular processes and is activated in both cancer and inflammatory cells. In hydroethanolic fruit extracts, 5.0–5.9% of total polyphenols were determined spectrophotometrically, including high amounts of hydroxycinnamic acid derivatives. HPLC analysis revealed that the black chokeberry and bilberry extracts contained 2.05 mg/g and 2.54 mg/g of chlorogenic acid, respectively. Using a time-resolved fluorescence resonance energy transfer (TR-FRET) assay, the extracts studied were found to have comparable inhibitory effects on Src tyrosine kinase, with IC₅₀ values of 366 µg/mL and 369 µg/mL, respectively. The results also indicated that chlorogenic acid contributes significantly to the observed effect. In addition, both fruit extracts exhibited antioxidant activity by scavenging DPPH and NO radicals with SC₅₀ values of 153–352 µg/mL. Our study suggested that black chokeberry and bilberry fruits may be beneficial in cancer and other inflammation-related diseases. Full article

Simufilam is a novel oral drug candidate in Phase 3 clinical trials for Alzheimer’s disease (AD) dementia. This small molecule binds an altered form of filamin A (FLNA) that occurs in AD. This drug action disrupts FLNA’s aberrant linkage to the α7 nicotinic acetylcholine receptor (α7nAChR), thereby blocking soluble amyloid beta_1–42 (Aβ₄₂)’s signaling via α7nAChR that hyperphosphorylates tau. Here, we aimed to clarify simufilam’s mechanism. We now show that simufilam reduced Aβ₄₂ binding to α7nAChR with a 10-picomolar IC₅₀ using time-resolved fluorescence resonance energy transfer (TR-FRET), a robust technology to detect highly sensitive molecular interactions. We also show that FLNA links to multiple inflammatory receptors in addition to Toll-like receptor 4 (TLR4) in postmortem human AD brains and in AD transgenic mice: TLR2, C-X-C chemokine receptor type 4 (CXCR4), C-C chemokine receptor type 5 (CCR5), and T-cell co-receptor cluster of differentiation 4 (CD4). These aberrant FLNA linkages, which can be induced in a healthy control brain by Aβ₄₂ incubation, were disrupted by simufilam. Simufilam reduced inflammatory cytokine release from Aβ₄₂-stimulated human astrocytes. In the AD transgenic mice, CCR5–G protein coupling was elevated, indicating persistent activation. Oral simufilam reduced both the FLNA–CCR5 linkage and the CCR5–G protein coupling in these mice, while restoring CCR5′s responsivity to C-C chemokine ligand 3 (CCL3). By disrupting aberrant FLNA–receptor interactions critical to AD pathogenic pathways, simufilam may promote brain health. Full article

SARS-CoV-2 exploits the homotrimer transmembrane Spike glycoproteins (S protein) during host cell invasion. The Omicron XBB subvariant, delta, and prototype SARS-CoV-2 receptor-binding domain show similar binding strength to hACE2 (human Angiotensin-Converting Enzyme 2). Here we utilized multiligand virtual screening to identify small molecule inhibitors for their efficacy against SARS-CoV-2 virus using QPLD, pseudovirus ACE2 Inhibition -Time Resolved Forster/Fluorescence energy transfer (TR-FRET) Assay Screening, and Molecular Dynamics simulations (MDS). Three hundred and fifty thousand compounds were screened against the macrodomain of the nonstructural protein 3 of SARS-CoV-2. Using TR-FRET Assay, we filtered out two of 10 compounds that had no reported activity in in vitro screen against Spike S1: ACE2 binding assay. The percentage inhibition at 30 µM was found to be 79% for “Compound F1877-0839” and 69% for “Compound F0470-0003”. This first of its kind study identified “FILLY” pocket in macrodomains. Our 200 ns MDS revealed stable binding poses of both leads. They can be used for further development of preclinical candidates. Full article

Monitoring the biological fate of medicaments within the environments of cancer cells is an important challenge which is nowadays the object of intensive studies. In this regard, rhodamine-based supramolecular systems are one of the most suitable probes used in drug delivery thanks to their high emission quantum yield and sensitivity to the environment which helps to track the medicament in real time. In this work, we used steady-state and time-resolved spectroscopy techniques to investigate the dynamics of the anticancer drug, topotecan (TPT), in water (pH ~6.2) in the presence of a rhodamine-labeled methylated β-cyclodextrin (RB-RM-βCD). A stable complex of 1:1 stoichiometry is formed with a K_eq value of ~4 × 10⁴ M⁻¹ at room temperature. The fluorescence signal of the caged TPT is reduced due to: (1) the CD confinement effect; and (2) a Förster resonance energy transfer (FRET) process from the trapped drug to the RB-RM-βCD occurring in ~43 ps with 40% efficiency. These findings provide additional knowledge about the spectroscopic and photodynamic interactions between drugs and fluorescent functionalized CDs, and may lead to the design of new fluorescent CD-based host–guest nanosystems with efficient FRET to be used in bioimaging for drug delivery monitoring. Full article

Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class. Full article