Fluorescence Polarization Research Articles

Formation of subwavelength tunable flat-top focus with double foci characteristic by tightly focused cylindrical vector beams

We report a numerical investigation on the generation of a subwavelength flat-top focus by tuning the polarization angle of the incident beam under tight focusing conditions. Our approach uses a binary phase mask containing two regions of opposite phases, with a variable diameter of the inner region. Properly tuning the diameter of the inner region of the mask, the flat-top profile of variable sizes (above and below the subwavelength region) is generated in a controlled manner with the various incident polarization angles of the beam and with the different numerical aperture (NA) value of the lens. Our study reveals that a particular cut-off value of polarization angle, above which the subwavelength flat-top is generated, corresponds to a specific NA that increases with the increase in the NA value. In addition, the emergence of the dual foci structure is observed in the longitudinal direction with increasing NA of the lens. The flat-top's polarization degrees-of-freedom and dual foci properties open up promising routes for potential applications in fluorescence polarization microscopy and multiplane particle trapping.

Modulating Interfacial Properties in Pseudoternary Microemulsions via Urea Addition: Impact of Cosurfactant on the Reverse Micellar Structure and Interactions.

We have studied the structural and interfacial properties of CTAB/isooctane/alcohol/aqueous urea reverse micelles (RMs) for the first time using time-resolved fluorescence and small-angle X-ray scattering techniques. The chain length of alcohol, used as cosurfactant, has been varied to design three microemulsion systems: CTAB/1-butanol, CTAB/1-hexanol, and CTAB/1-octanol/isooctane/water, at a fixed water loading ratio, w0 = 12. Time-resolved fluorescence anisotropy studies indicate that urea induces micellar aggregation in CTAB/1-butanol and CTAB/1-hexanol RMs but breaks down RM aggregates in CTAB/1-octanol RMs. Urea addition slows down solvation dynamics inside RMs at higher urea concentrations, evident from the longer lifetimes of solvent correlation decay. The underlying changes in microemulsion structure and intermicellar interactions are studied using small-angle X-ray scattering studies. The significant intermicellar interactions were modeled using the sticky hard sphere (SHS) for the CTAB/1-butanol and CTAB/1-hexanol RMs and by using the Macroion model for the CTAB/1-octanol RMs. The two different structural factors highlight the dominance of attractive and repulsive forces, respectively. Although there is no change in RM shape, the combination of urea addition and chain length variation in cosurfactants significantly alters the size and interface in these pseudoternary RMs.

Insights into the Interaction Landscape of the EVH1 Domain of Mena.

The Enabled/VASP homology 1 (EVH1) domain is a small module that interacts with proline-rich stretches in its ligands and is found in various signaling and scaffolding proteins. Mena, the mammalian homologue of Ena, is involved in diverse actin-associated events, such as membrane dynamics, bacterial motility, and tumor intravasation and extravasation. Two-dimensional (2D) 1H-15N HSQC NMR was used to study Mena EVH1 binding properties, defining the amino acids involved in ligand recognition for the physiological ligands ActA and PCARE, and a synthetic polyproline-inspired small molecule (hereafter inhibitor 6c). Chemical shift perturbations indicated that proline-rich segments bind in the conserved EVH1 hydrophobic cleft. The PCARE-derived peptide elicited more perturbations compared to the ActA-derived peptide, consistent with a previous report of a structural alteration in the solvent-exposed β7-β8 loop. Unexpectedly, EVH1 and the proline-rich segment of PTP1B did not exhibit NMR chemical shift perturbations; however, the high-resolution crystal structure implicated the conserved EVH1 hydrophobic cleft in ligand recognition. Intrinsic steady-state fluorescence and fluorescence polarization assays indicate that residues outside the proline-rich segment enhance the ligand affinity for EVH1 (Kd = 3-8 μM). Inhibitor 6c displayed tighter binding (Kd ∼ 0.3 μM) and occupies the same EVH1 cleft as physiological ligands. These studies revealed that the EVH1 domain enhances ligand affinity through recognition of residues flanking the proline-rich segments. Additionally, a synthetic inhibitor binds more tightly to the EVH1 domain than natural ligands, occupying the same hydrophobic cleft.

OOPS: Object-Oriented Polarization Software for analysis of fluorescence polarization microscopy images.

Most essential cellular functions are performed by proteins assembled into larger complexes. Fluorescence Polarization Microscopy (FPM) is a powerful technique that goes beyond traditional imaging methods by allowing researchers to measure not only the localization of proteins within cells, but also their orientation or alignment within complexes or cellular structures. FPM can be easily integrated into standard widefield microscopes with the addition of a polarization modulator. However, the extensive image processing and analysis required to interpret the data have limited its widespread adoption. To overcome these challenges and enhance accessibility, we introduce OOPS (Object-Oriented Polarization Software), a MATLAB package for object-based analysis of FPM data. By combining flexible image segmentation and novel object-based analyses with a high-throughput FPM processing pipeline, OOPS empowers researchers to simultaneously study molecular order and orientation in individual biological structures; conduct population assessments based on morphological features, intensity statistics, and FPM measurements; and create publication-quality visualizations, all within a user-friendly graphical interface. Here, we demonstrate the power and versatility of our approach by applying OOPS to punctate and filamentous structures.

Discovering New Metallo-Deubiquitinase CSN5 Inhibitors by a Non-Catalytic Activity Assay Platform.

COP9 signalosome catalytic subunit CSN5 plays a key role in tumorigenesis and tumor immunity, showing potential as an anticancer target. Currently, only a few CSN5 inhibitors have been reported, at least partially, due to the challenges in establishing assays for CSN5 deubiquitinase activity. Here, we present the establishment and validation of a simple and reliable non-catalytic activity assay platform for identifying CSN5 inhibitors utilizing a new fluorescent probe, CFP-1, that exhibits enhanced fluorescence and fluorescence polarization features upon binding to CSN5. By using this platform, we identified 2-aminothiazole-4-carboxylic acids as new CSN5 inhibitors, which inhibited CSN5 but slightly downregulated PD-L1 in cancer cells. Furthermore, through the integration of deep learning-enabled virtual screening, we discovered that shikonins are nanomolar CSN5 inhibitors, which can upregulate PD-L1 in HCT116 cells. The binding modes of these structurally distinct inhibitors with CSN5 were explored by using microsecond-scale molecular dynamics simulations and tryptophan quenching assays.

(Invited) Comprehensive Analysis of Exciton Diffusion with Time-Resolved Fluorescence Spectroscopy, Anisotropy and Imaging

Excitons produced in materials play a crucial role in photo-energy conversion such as photovoltaics and electroluminescence devices. The temporal and spatial diffusion of excitons is a key factor for dominating the fundamental performance of optoelectronic materials. In this context, analysis of the exciton diffusion dynamics is an important issue for obtaining the rational design of the materials. Excitons diffusing in materials can be characterized by several parameters such as the energy level, orientation of the transition dipole moment and spatial distribution, and the detection of these properties is required for the comprehensive analysis. In the present study, we show time-resolved fluorescence methods for detecting the above relevant properties of excitons.First, time-resolved fluorescence spectroscopy is a basic technique for analyzing the exciton diffusion dynamics. Fluorescence intensity is detected as functions of observation wavelength and time, and the spectral intensity and shift of fluorescence bands provide information on the population of excitons and their energy level. For example, dynamic Stokes shift indicates downhill energy migration and excitons are trapped by the defective site in molecular aggregates. Second, fluorescence anisotropy can be a good indicator for tracking the exciton diffusion in amorphous materials. Fluorescence anisotropy is sensitive to orientation change in transition dipole moments (TDMs) between absorption and fluorescence. In systems where the relative orientation of adjacent molecules is different from each other, such as amorphous solids, the exciton diffusion is accompanied with the change in TDM. Thus, appropriate modeling of molecular alignment enables quantitative estimation of the exciton diffusion coefficient and diffusion length from the anisotropy signal. Finally, time-resolved imaging is a combination of time-resolved spectroscopy and super-resolution microscopy, and an emergent technique for visualizing real-space propagation of excitons in materials. The diffraction-limited excitation beam produces excitons in several hundreds of nanometers and the subsequent exciton diffusion is evaluated by the spatial width of the fluorescence spot. The temporal broadening of the fluorescence spot directly reflects the exciton distribution and we can intuitively obtain the diffusion coefficient and length. Unlike the conventional methods for bulk materials, the advantage of the time-resolved imaging is the applicability to materials with nano- and meso-scale heterogeneous structure and it enables site-selective evaluation of the exciton transport capability. In the conference site, we will also show the application of the above methods to supramolecular polymers and thermally activated delayed fluorescence materials. Figure 1

Identification of novel bromodomain inhibitors of Trypanosoma cruzi bromodomain factor 2 (TcBDF2) using a fluorescence polarization-based high-throughput assay.

Bromodomains are structural folds present in all eukaryotic cells that bind to other proteins recognizing acetylated lysines. Most proteins with bromodomains are part of nuclear complexes that interact with acetylated histone residues and regulate DNA replication, transcription, and repair through chromatin structure remodeling. Bromodomain inhibitors are small molecules that bind to the hydrophobic pocket of bromodomains, interfering with the interaction with acetylated histones. Using a fluorescent probe, we have developed an assay to select inhibitors of the bromodomain factor 2 of Trypanosoma cruzi (TcBDF2) using fluorescence polarization. Initially, a library of 28,251 compounds was screened in an endpoint assay. The top 350-ranked compounds were further analyzed in a dose-response assay. From this analysis, seven compounds were obtained that had not been previously characterized as bromodomain inhibitors. Although these compounds did not exhibit significant trypanocidal activity, all showed bona fide interaction with TcBDF2 with dissociation constants between 1 and 3 µM validating these assays to search for bromodomain inhibitors.

Photophysics in Biomembranes: Computational Insight into the Interaction between Lipid Bilayers and Chromophores.

ConspectusLight is ubiquitously available to probe the structure and dynamics of biomolecules and biological tissues. Generally, this cannot be done directly with visible light, because of the absence of absorption by those biomolecules. This problem can be overcome by incorporating organic molecules (chromophores) that show an optical response in the vicinity of those biomolecules. Since those optical properties are strongly dependent on the chromophore's environment, time-resolved spectroscopic studies can provide a wealth of information on biosystems at the molecular scale in a nondestructive way. In this work, we give an overview on the multiscale computational strategy developed by us in the last eight years and prove that theoretical studies and simulations are needed to explain, guide, and predict observations in fluorescence experiments. As we challenge the accepted views on existing probes, we discover unexplored abilities that can discriminate surrounding lipid bilayers and their temperature-dependent as well as solvent-dependent properties. We focus on three archetypal chromophores: diphenylhexatriene (DPH), Laurdan, and azobenzene. Our method shows that conformational changes should not be neglected for the prototype rod-shaped molecule DPH. They determine its position and orientation in a liquid-ordered (Lo) sphingomyelin/cholesterol (SM/Chol) bilayer and are responsible for a strong differentiation of its absorption spectra and fluorescence decay times in dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) membranes, which are at room temperature in liquid-disordered (Ld) and solid-gel (So) phases, respectively. Thanks to its pronounced first excited state dipole moment, Laurdan has long been known as a solvatochromic probe. Since this molecule has however two conformers, we prove that they exhibit different properties in different lipid membrane phases. We see that the two conformers are only blocked in one phase but not in another. Supported by fluorescence anisotropy decay simulations, Laurdan can therefore be regarded as a molecular rotor. Finally, the conformational versatility of azobenzene in saturated Ld lipid bilayers is simulated, along with its photoisomerization pathways. By means of nonadiabatic QM/MM surface hopping analyses (QM/MM-SH), a dual mechanism is found with a torsional mechanism and a slow conversion for trans-to-cis. For cis-to-trans, simulations show a much higher quantum yield and a so-called "pedal-like" mechanism. The differences are related to the different potential energy surfaces as well as the interactions with the surrounding alkyl chains. When tails of increased length are attached to this probe, cis is pushed toward the polar surface, while trans is pulled toward the center of the membrane.

Juglone suppresses vasculogenic mimicry in glioma through inhibition of HuR-mediated VEGF-A expression

Vasculogenic mimicry (VM) serves as a vascular-like channel that provides important substances for tumor growth and is a primary factor in glioblastoma (GBM) drug resistance. Human Antigen R (HuR)—an mRNA-binding protein—is highly expressed in GBM, closely related to tumor progression, and deemed a potential drug target. Although some small-molecule compounds have been identified to disrupt HuR binding to target mRNA, they remain in the preclinical research stage, suggesting the need for further validation and development of HuR inhibitors. In our study, we aim to screen for potential HuR inhibitors and investigate their efficacy and molecular mechanisms in GBM. We employed the fluorescence polarization method to identify HuR inhibitors from a natural compound library, confirming the efficacy of juglone in effectively inhibiting the binding of HuR to AREVegf-a. Further validation of the binding of juglone to HuR at the protein level was conducted through electrophoretic mobility shift analysis, surface plasmon resonance, and molecular docking. Furthermore, juglone demonstrated inhibitory effects on glioma growth and VM formation in vitro and in vivo. Moreover, it was observed that juglone reversed epithelial-mesenchymal transition by inhibiting the VEGF-A/VEGFR2/AKT/SNAIL signaling pathway. Finally, we established the capability of juglone to target HuR in U251 cells through HuR knockdown, mRNA stability, and cell thermal shift assays. Therefore, this study identifies juglone as a novel HuR inhibitor, potentially offering promise as a lead compound for anti-VM therapy in GBM by targeting HuR.Abbreviations: AKT, protein kinase B; ARE, adenine-and uridine-rich elements; CETSA, cellular thermal shift assay; DMEM, Dulbecco’s modified Eagle’s medium; ELISA, enzyme linked immune sorbent assay; EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; FP, fluorescence polarization; GBM, glioblastoma; HTS, high-throughput screening; HuR, human antigen R; IF, Immunofluorescence; PAS, periodic acid-Schiff; PI3K, phosphoinositide-3 kinase; qRT-PCR, quantitative real-time PCR; RRMs, RNA recognition motifs; SPR, surface plasmon resonance. TMZ, temozolomide; VM, vasculogenic mimicry; VEGF-A, Vascular endothelial growth factor-A; VEGFR2, Vascular endothelial growth factor receptor-2.

Novel GPIb-independent platelet aggregation induced by botrocetin: implications for diagnosis and antithrombotic therapy

BackgroundSnake venom botrocetin facilitates von Willebrand factor (VWF) binding to platelet GPIbα and has been widely used for the diagnosis of von Willebrand disease and GPIb-related disorders. Botrocetin is also commonly employed for the development/characterization of antithrombotics targeting the GPIb-VWF axis. ObjectivesTo explore the alternative receptor(s)/mechanisms that participate in botrocetin-induced platelet aggregation. MethodsThe effects of botrocetin on platelet aggregation were examined using platelets from wild-type, VWF- and fibrinogen-deficient, GPIbα-deficient, IL4Rα/GPIbα-transgenic and αIIbβ3-deficient mice and Bernard–Soulier syndrome and healthy human samples. Platelet-fibrinogen and platelet-VWF interaction were measured using flow cytometry. GPIbα-VWF binding was evaluated utilizing enzyme-linked immunosorbent assay. Botrocetin-αIIbβ3 and botrocetin-GPIbα interactions were measured using enzyme-linked immunosorbent assay and fluorescence anisotropy assays. Heparinized whole blood from healthy donors was examined for thrombus formation and growth in a perfusion chamber. ResultsBotrocetin could induce aggregation of platelets from a Bernard–Soulier syndrome patient and GPIbα-deficient mice as well as platelets lacking the N-terminal extracellular domain of GPIbα. Botrocetin could interact with αIIbβ3 and facilitated αIIbβ3-VWF interaction independent of GPIb. Botrocetin competitively bound to the ligand-binding domain of activated rather than resting αIIbβ3. Although botrocetin-induced platelet aggregation requires VWF, strikingly, in the absence of VWF, botrocetin blocked fibrinogen and other ligand binding to αIIbβ3 and inhibited platelet aggregation and thrombus formation. Consistently, recombinant botrocetin defective in VWF binding inhibited αIIbβ3- and GPIb-mediated platelet aggregation, spreading, and thrombus formation. ConclusionOur study provides insights into avoiding the misdiagnosis of GPIb-related disorders and developing botrocetin mutants as potential new antithrombotics that may simultaneously target both αIIbβ3 and GPIbα.

Room temperature luminescence of 1,N2-etheno-2-aminopurine in poly (vinyl alcohol) films.

We studied spectral properties of 1,N2-etheno-2-aminopurine after immobilization in poly (vinyl alcohol) films. The absorption spectrum of 1,N2-ε2APu consists of two peaks centered at 300 and 370 nm, and the fluorescence spectrum has maximum at about 460 nm. The fluorescence quantum efficiency is 62%. The fluorescence anisotropy reaches a value of 0.3 at longer wavelengths, while it is low at shorter wavelengths (corresponding to the second single excited state). The 1,N2-ε2APu has a relatively long fluorescence lifetime of about 16 ns and a noticeable room temperature phosphorescence with a lifetime of about 220 ms. A broad phosphorescence emission band (425-675 nm) is centered at about 530 nm and markedly overlaps with fluorescence at shorter wavelengths. Surprisingly, the phosphorescence excitation spectrum of 1,N2-ε2APu-doped poly (vinyl alcohol) film differs from the absorption and fluorescence excitation spectra. The strongest room temperature phosphorescence excitation is about 335 nm. At longer excitation wavelengths, above 450 nm, where fluorescence cannot be excited, a triplet excitation is still possible. The 1,N2-ε2APu phosphorescence anisotropy spectra confirm direct triplet state excitation. The ability to excite molecules at long wavelengths can find applications in the study of biological molecules that are unstable when excited at high energies.

Unraveling the structure and function of a novel SegC protein interacting with the SegAB chromosome segregation complex in Archaea.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

The many faces of brucellosis: diagnostic and management approach.

This review aims to highlight the multifaceted nature of brucellosis, emphasizing the latest advancements in its diagnosis and management. Given the global prevalence and potential complications of brucellosis, understanding recent advancements in diagnostic techniques and treatment strategies is crucial for clinicians. Recent literature reveals significant progress in diagnostic methods, including the application of fluorescence polarization immunoassay and time-resolved fluorescence resonance energy transfer technologies as well as the invention of artificial Brucella antigens, which offer enhanced sensitivity and specificity. Advances in molecular diagnostics and serological tests have improved early detection rates, however their interpretation remains challenging. Evolving treatment regimens such as the use of hydroxychloroquine as part of triple therapy and the use of nano-delivery systems in therapies have shown promise, in hopes of reducing relapse rates and managing chronic cases. The findings underscore the necessity for clinicians to adopt a comprehensive approach to diagnosing and managing brucellosis. Integrating advanced diagnostic tools with tailored therapeutic strategies can significantly improve patient outcomes. Future research should focus on optimizing these diagnostic techniques and exploring novel therapeutic agents.

Elucidating the potential of isorhapontigenin in targeting the MgrA regulatory network: a paradigm shift for attenuating MRSA virulence.

As methicillin-resistant Staphylococcus aureus (MRSA) exhibits formidable resistance to many drugs, the imperative for alternative therapeutic strategies becomes increasingly evident. At the heart of our study is the identification of a novel inhibitor through fluorescence anisotropy assays, specifically targeting the crucial multiple gene regulator A (MgrA) regulatory network in S. aureus. Isorhapontigenin (Iso), a natural compound, exhibits outstanding inhibitory efficacy, modulating bacterial virulence pathways without exerting direct bactericidal activity. This suggests a paradigm shift toward attenuating virulence instead of purely focusing on bacterial elimination. Through comprehensive in vitro and in vivo evaluations, we elucidated the complex interplay between Iso and MgrA, leading to reduced S. aureus adhesion, and overall virulence. At the cellular level, Iso offers significant protection to A549 cells infected with S. aureus, reducing cellular damage. Importantly, Iso augments the chemotaxis of neutrophils, curtailing the immune evasion capabilities of S. aureus. Furthermore, in vivo investigations highlight the notable effectiveness of Iso against MRSA-induced pneumonia and within the Galleria mellonella infection model, underscoring its pivotal role in the evolving realm of antibacterial drug discovery. Significantly, when Iso is used in combination with vancomycin, it outperforms its solo application, indicating a more pronounced therapeutic impact. This seminal research emphasizes Iso's potential as a primary defense against the surge of multidrug-resistant pathogens, heralding new prospects in antimicrobial therapy.

Click chemistry-based fluorescence polarization sensor for sensitive detection of ampicillin

Ampicillin (AMP) is a β-lactam antibiotic that can inhibit bacterial wall synthesis. The overuse and misuse of AMP makes it micropollutant that commonly found in food and various environmental media. In this work, a fluorescence polarization sensor was designed to sensitive detection of trace ampicillin based on click chemistry, using graphene oxide (GO) as a fluorescence polarization (FP) signal enhancement element. First, when ampicillin binds to its aptamer (apt), the adjacent alkyne and azide groups are separated, hindering the click-linking reaction. When Carboxyfluorescein (FAM) fluorophore-labeled probe (C-FAM) is added, its protruding 3-terminal FAM is recognized and cleaved by exonuclease I (EXO I), releasing fluorophores free that could not be adsorbed on GO, resulting in a lo0wer polarization signal. If there is no AMP in the system, aptamer probe is connected to its complementary chain ends by a click reaction. After C-FAM hybridizes with apt, the apt/P duplex is opened and the prominent single-stranded ends adsorb on the GO, leading a significantly enhanced FP signal. According to the relationship between the difference in FP values and the concentrations of AMP, the limit of detection of proposed method is as low as 80 pg/mL. This assay has a wide linear range plus excellent selectivity, and has been applied to detect AMP in milk and river water samples with satisfactory results, which demonstrates that the FP sensor has great potential for practical applications in food safety and environmental protection fields.

Hydration- and Temperature-Dependent Fluorescence Spectra of Laurdan Conformers in a DPPC Membrane.

The widely used Laurdan probe has two conformers, resulting in different optical properties when embedded in a lipid bilayer membrane, as demonstrated by our previous simulations. Up to now, the two conformers' optical responses have, however, not been investigated when the temperature and the phase of the membrane change. Since Laurdan is known to be both a molecular rotor and a solvatochromic probe, it is subject to a profound interaction with both neighboring lipids and water molecules. In the current study, molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics calculations are performed for a DPPC membrane at eight temperatures between 270K and 320K, while the position, orientation, fluorescence lifetime and fluorescence anisotropy of the embedded probes are monitored. The importance of both conformers is proven through a stringent comparison with experiments, which corroborates the theoretical findings. It is seen that for Conf-I, the excited state lifetime is longer than the relaxation of the environment, while for Conf-II, the surroundings are not yet adapted when the probe returns to the ground state. Throughout the temperature range, the lifetime and anisotropy decay curves can be used to identify the different membrane phases. The current work might, therefore, be of importance for biomedical studies on diseases, which are associated with cell membrane transformations.

Using a static magnetic field to attenuate the severity in COVID-19-invaded lungs

Two important factors affecting the progress of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the S-protein binding function of ACE2 receptors and the membrane fluidity of host cells. This study aimed to evaluate the effect of static magnetic field (SMF) on S-protein/ACE2 binding and cellular membrane fluidity of lung cells, and was performed in vitro using a Calu-3 cell model and in vivo using an animal model. The ability of ACE2 receptors to bind to SARS-CoV-2 spike protein on host cell surfaces under SMF stimulation was evaluated using fluorescence images. Host lung cell membrane fluidity was tested using fluorescence polarization to determine the effects of SMF. Our results indicate that 0.4 T SMF can affect binding between S-protein and ACE2 receptors and increase Calu-3 cell membrane fluidity, and that SMF exposure attenuates LPS-induced alveolar wall thickening in mice. These results may be of value for developing future non-contact, non-invasive, and low side-effect treatments to reduce disease severity in COVID-19-invaded lungs.

Deep learning-based cell segmentation for rapid optical cytopathology of thyroid cancer

Fluorescence polarization (Fpol) imaging of methylene blue (MB) is a promising quantitative approach to thyroid cancer detection. Clinical translation of MB Fpol technology requires reduction of the data analysis time that can be achieved via deep learning-based automated cell segmentation with a 2D U-Net convolutional neural network. The model was trained and tested using images of pathologically diverse human thyroid cells and evaluated by comparing the number of cells selected, segmented areas, and Fpol values obtained using automated (AU) and manual (MA) data processing methods. Overall, the model segmented 15.8% more cells than the human operator. Differences in AU and MA segmented cell areas varied between − 55.2 and + 31.0%, whereas differences in Fpol values varied from − 20.7 and + 10.7%. No statistically significant differences between AU and MA derived Fpol data were observed. The largest differences in Fpol values correlated with greatest discrepancies in AU versus MA segmented cell areas. Time required for auto-processing was reduced to 10 s versus one hour required for MA data processing. Implementation of the automated cell analysis makes quantitative fluorescence polarization-based diagnosis clinically feasible.

The Role of Surfactant Head Group Charge on Binding of Hoechst 33258 with Micelles

AbstractOwing to the diverse biological application, photophysical behaviour of Hoechst 33258 (H33258) is an important topic in biophysical research. Here, we have reported the photophysical behavior of H33258 in model biomembrane mimicking micellar microenvironment. In order to understand the molecular mechanism of the interaction of H33258 with micelles, we have used cationic (Hexadecetyltrimethyl ammonium bromide (CTAB)), anionic (sodium dodecyl sulphate (SDS)), and non‐ionic (t‐octylphenoxypolyoxyethanol, Triton‐X100 (TX 100)) micelles. From the steady‐state emission study the evaluated apparent binding constant (Kb) values for the complex formation between H33258 and CTAB, SDS and TX 100 micelle were (1.45±0.42)×105, (31.95±4.89)×105 and (3.10±0.73)×105 M−1, respectively. Thus, it was found that the Kb value was greater in the case of anionic micelle than non‐ionic and cationic micelles. Such differences in Kb values were due to the electrostatic interaction and hydrogen bond formation between the probe molecule and micelles. In addition, time‐resolved fluorescence decay and time‐resolved fluorescence anisotropy studies were performed to understand the dynamical behavior of H33258 in presence of micelles. The interaction of H33258 with micelles might be useful to study the structure and dynamics of different bio‐membranes and to formulate new drug delivery system.

The RNA-binding Selectivity of the RGG/RG Motifs of hnRNP U is Abolished by Elements Within the C-terminal Intrinsically Disordered Region

The abundant nuclear protein hnRNP U interacts with a broad array of RNAs along with DNA and protein to regulate nuclear chromatin architecture. The RNA-binding activity is achieved via a disordered ∼100 residue C-terminal RNA-binding domain (RBD) containing two distinct RGG/RG motifs. Although the RNA-binding capabilities of RGG/RG motifs have been widely reported, less is known about hnRNP U’s RNA-binding selectivity. Furthermore, while it is well established that hnRNP U binds numerous nuclear RNAs, it remains unknown whether it selectively recognizes sequence or structural motifs in target RNAs. To address this question, we performed equilibrium binding assays using fluorescence anisotropy (FA) and electrophoretic mobility shift assays (EMSAs) to quantitatively assess the ability of human hnRNP U RBD to interact with segments of cellular RNAs identified from eCLIP data. These RNAs often, but not exclusively, contain poly-uridine or 5′-AGGGAG sequence motifs. Detailed binding analysis of several target RNAs reveal that the hnRNP U RBD binds RNA in a promiscuous manner with high affinity for a broad range of structured RNAs, but with little preference for any distinct sequence motif. In contrast, the isolated RGG/RG of hnRNP U motif exhibits a strong preference for G-quadruplexes, similar to that observed for other RGG motif bearing peptides. These data reveal that the hnRNP U RBD attenuates the RNA binding selectivity of its core RGG motifs to achieve an extensive RNA interactome. We propose that a critical role of RGG/RG motifs in RNA biology is to alter binding affinity or selectivity of adjacent RNA-binding domains.