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We combine on-the-fly trajectory surface hopping simulations and the doorway–window representation of nonlinear optical response functions to create an efficient protocol for the evaluation of time- and frequency-resolved fluorescence (TFRF) spectra and anisotropies of the realistic polyatomic systems. This approach gives the effective description of the proper (e.g., experimental) pulse envelopes, laser field polarizations, and the proper orientational averaging of TFRF signals directly from the well-established on-the-fly nonadiabatic dynamic simulations without extra computational cost. To discuss the implementation details of the developed protocol, we chose cis-azobenzene as a prototype to simulate the time evolution of the TFRF spectra governed by its nonadiabatic dynamics. The results show that the TFRF is determined by the interplay of several key factors, i.e., decays of excited-state populations, evolution of the transition dipole moments along with the dynamic propagation, and scaling factor of the TFRF signals associated with the cube of emission frequency. This work not only provides an efficient and effective approach to simulate the TFRF and anisotropies of realistic polyatomic systems but also discusses the important relationship between the TFRF signals and the underlining nonadiabatic dynamics. [ABSTRACT FROM AUTHOR]

The excessive cosolute densities in the intracellular fluid create a physicochemical condition called macromolecular crowding (MMC). Intracellular MMC entropically maintains the biochemical thermodynamic equilibria by favoring associative reactions while hindering transport processes. Rapid cell volume shrinkage during extracellular hypertonicity elevates the MMC and disrupts the equilibria, potentially ushering cell death. Consequently, cells actively counter the hypertonic stress through regulatory volume increase (RVI) and restore the MMC homeostasis. Here, we establish fluorescence anisotropy of EGFP as a reliable tool for studying cellular MMC and explore the spatiotemporal dynamics of MMC during cell volume instabilities under multiple conditions. Our studies reveal that the actin cytoskeleton enforces spatially varying MMC levels inside adhered cells. Within cell populations, MMC is uncorrelated with nuclear DNA content but anti-correlated with the cell spread area. Although different cell lines have statistically similar MMC distributions, their responses to extracellular hypertonicity vary. The intensity of the extracellular hypertonicity determines a cell's ability for RVI, which correlates with nuclear factor kappa beta (NFkB) activation. Pharmacological inhibition and knockdown experiments reveal that tumor necrosis factor receptor 1 (TNFR1) initiates the hypertonicity-induced NFkB signaling and RVI. At severe hypertonicities, the elevated MMC amplifies cytoplasmic microviscosity and hinders receptor interacting protein kinase 1 (RIPK1) recruitment at the TNFR1 complex, incapacitating the TNFR1-NFkB signaling and consequently, RVI. Together, our studies unveil the involvement of TNFR1-NFkB signaling in modulating RVI and demonstrate the pivotal role of MMC in determining cellular osmoadaptability. [ABSTRACT FROM AUTHOR]

In this paper, novel tellurate crystals Lu2TeO6 and Nd:Lu2TeO6 in a trigonal system are successfully grown by the top-seeded solution growth (TSSG) method using TeO2 and Li2CO3 as fluxes. The full-width at half maximum of the X-ray diffraction rocking curve of the Lu2TeO6(100) and (001) wafers are determined to be 37′′ and 47′′, respectively. The transmittance spectra suggest that the Lu2TeO6 crystal has a wide transparency window from 0.5 to 6.5 μm. The Lu2TeO6 crystal exhibits excellent thermal properties with thermal conductivity coefficients of 2.709 W m−1 K−1 and 3.782 W m−1 K−1 along the a and c axes at 50 °C, respectively. Although the Nd:Lu2TeO6 crystal shows small polarized absorption and fluorescence anisotropy with absorption and emission peaks at 808 and 1064 nm, both the absorption and emission peak intensities along the c-axis are significantly higher compared to the a-axis. The spectra and thermal properties indicate that the Lu2TeO6 crystal may be a potential laser crystal. [ABSTRACT FROM AUTHOR]

An assembly of metal nanoclusters driven by appropriate surface ligands and solvent environment may engender entirely new photoluminescence (PL). Herein, we first synthesize histidine (His) stabilized copper nanoparticles (CuNPs) and, subsequently, copper nanoclusters (CuNCs) from it using 3‐mercaptopropionic acid (MPA) as an etchant. The CuNCs originally emit bluish‐green (λem=470 nm) PL with a low quantum yield (QY∼1.8 %). However, it transformed into a dual‐emissive nanocluster assembly (Zn‐CuNCs) in the presence of Zn(II) salt, having a distinct blue emission band (λem=420 nm) and a red emission band (λem=615 nm) with eight times QY (∼9.1 %) enhancement. The temperature‐dependent emission spectra of Zn‐CuNCs depicted that the blue emission band persists for all the temperature ranges (0–80 °C) while the red emission band vanishes at high temperatures (70–80 °C). Thus, the blue emission may originate from the locally excited state (LES) emission of the nanoclusters, while the red emission originates from through‐space interaction (TSI) and Cu(I)...Cu(I) interaction within the assembly. Adding dimethyl sulfoxide (DMSO) further modifies the emission intensities; the red band was amplified four times, while the blue band was diminished by 2.5 times. The transmission electron microscopy (TEM) images unveiled that the Zn‐CuNCs are a large assembly of tiny nanoclusters, which become more compact in DMSO. The blue emission possesses steady‐state fluorescence anisotropy, while the red emission shows no anisotropy. Further, near‐perfect white light emission(WLE) was rendered with CIE coordinates of (0.33, 0.32) by combining the dual emission of the Zn‐CuNCs with the original green emission of the CuNCs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thiamine (vitamin B1) is essential for glucose catabolism. In the yeast species, Nakaseomyces glabratus (formerly Candida glabrata) and Saccharomyces cerevisiae , the transcription factor Pdc2 (with Thi3 and Thi2) upregulates pyruvate decarboxylase (PDC) genes and thiamine biosynthetic and acquisition (THI) genes during starvation. There have not been genome-wide analyses of Pdc2 binding. Previously, we identified small regions of Pdc2-regulated genes sufficient to confer thiamine regulation. Here, we performed deletion analyses on these regions. We observed that when the S. cerevisiae PDC5 promoter is introduced into N. glabratus , it is thiamine starvation inducible but does not require the Thi3 coregulator. The ScPDC5 promoter contains a 22-bp duplication with an AT-rich spacer between the 2 repeats, which are important for regulation. Loss of the first 22-bp element does not eliminate regulation, but the promoter becomes Thi3 dependent, suggesting cis architecture can generate a Thi3-independent, thiamine starvation inducible response. Whereas many THI promoters only have 1 copy of this element, addition of the first 22-bp element to a Thi3-dependent promoter confers Thi3 independence. Finally, we performed fluorescence anisotropy and chromatin immunoprecipitation sequencing. Pdc2 and Thi3 bind to regions that share similarity to the 22-bp element in the ScPDC5 promoter and previously identified cis elements in N. glabratus promoters. Also, while Pdc2 binds to THI and PDC promoters, neither Pdc2 nor Thi3 appears to bind the evolutionarily new NgPMU3 promoter that is regulated by Pdc2. Further study is warranted because PMU3 is required for cells to acquire thiamine from environments where thiamine is phosphorylated, such as in the human bloodstream. [ABSTRACT FROM AUTHOR]

Owing 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. [ABSTRACT FROM AUTHOR]

Forkhead box P2 (FOXP2) regulates expression of various genes and is associated with language, speech and neural development as well as cancer. Since there may be a putative link between sex and language and because transcription factors rarely function in isolation, this study aims to investigate whether FOXP2 directly associates with oestrogen receptor α (ER1), a nuclear receptor responsible for sexual differentiation that is also associated with cancer. Isothermal titration calorimetry and fluorescence anisotropy were used to investigate the interaction between the DNA-binding forkhead domain (FHD) of FOXP2, the N-terminal region (NT) of FOXP2, and the ligand-binding domain (LBD) of ER1. ER1 LBD does not interact with FOXP2 NT but associates with apo-FOXP2 FHD in an enthalpically favourable manner. The affinity of this interaction is inversely correlated to the salt concentration. Additionally, FOXP2 FHD that is bound to ER1 LBD, has reduced ability to interact with its cognate DNA. This research identifies a novel interaction between ER1 LBD and FOXP2 FHD and shows that the interaction is regulated by salt. Moreover, FOXP2 FHD cannot bind to both ER1 LBD and DNA simultaneously, suggesting that this interaction could be involved in regulating the transcriptional pathway of FOXP2 should the interaction be found in vivo. This study could serve as a foundation for uncovering the basis of sexual dimorphism in speech and language development and related disorders and potentially offers an alternate for targeted cancer therapies. • A novel interaction is identified in vitro between FOXP2 FHD and ER1 LBD. • This interaction has both an electrostatic and hydrophobic interface and involves the DNA-binding Helix 3 of FOXP2 FHD. • The ligand binding region and the LXXLL-binding AF-2 region of ER1 LBD is not involved in the protein-protein interaction. • FOXP2 FHD can bind either to DNA or to ER1 but not to both simultaneously. • The identified FOXP2-ER1 interaction could be involved in regulating FOXP2's transcriptional activity. [ABSTRACT FROM AUTHOR]

Lipid oxidation is a major cause of quality deterioration that decreases the shelf-life of muscle-based foods (red meat, poultry, and fish), in which heme proteins, particularly hemoglobin and myoglobin, are the primary pro-oxidants. Due to increasing consumer concerns over synthetic chemicals, extensive research has been carried out on natural antioxidants, especially plant polyphenols. The conventional opinion suggests that polyphenols inhibit lipid oxidation of muscle foods primarily owing to their strong hydrogen-donating and transition metal-chelating activities. Recent developments in analytical techniques (e.g., protein crystallography, nuclear magnetic resonance spectroscopy, fluorescence anisotropy, and molecular docking simulation) allow deeper understanding of the molecular interaction of polyphenols with heme proteins, phospholipid membrane, reactive oxygen species, and reactive carbonyl species; hence, novel hypotheses regarding their antioxidant mechanisms have been formulated. In this review, we summarize five direct and three indirect pathways by which polyphenols inhibit heme protein-mediated lipid oxidation in muscle foods. We also discuss the relation between chemical structures and functions of polyphenols as antioxidants. [ABSTRACT FROM AUTHOR]

Fluorescence induced by the excitation of a fluorophore with plane-polarized light has a different polarization depending on the size of the fluorophore-containing reagent and the rate of its rotation. Based on this effect, many analytical systems have been implemented in which an analyte contained in a sample and labeled with a fluorophore (usually fluorescein) competes to bind to antibodies. Replacing antibodies in such assays with aptamers, low-cost and stable oligonucleotide receptors, is complicated because binding a fluorophore to them causes a less significant change in the polarization of emissions. This work proposes and characterizes the compounds of the reaction medium that improve analyte binding and reduce the mobility of the aptamer–fluorophore complex, providing a higher analytical signal and a lower detection limit. This study was conducted on aflatoxin B1 (AFB1), a ubiquitous toxicant contaminating foods of plant origins. Eight aptamers specific to AFB1 with the same binding site and different regions stabilizing their structures were compared for affinity, based on which the aptamer with 38 nucleotides in length was selected. The polymers that interact reversibly with oligonucleotides, such as poly-L-lysine and polyethylene glycol, were tested. It was found that they provide the desired reduction in the depolarization of emitted light as well as high concentrations of magnesium cations. In the selected optimal medium, AFB1 detection reached a limit of 1 ng/mL, which was 12 times lower than in the tris buffer commonly used for anti-AFB1 aptamers. The assay time was 30 min. This method is suitable for controlling almond samples according to the maximum permissible levels of their contamination by AFB1. The proposed approach could be applied to improve other aptamer-based analytical systems. [ABSTRACT FROM AUTHOR]

We present a triple-mode nanosensor platform for nucleic acid detection utilizing fluorescence anisotropy and Förster resonance energy transfer (FRET) strategies. The self-assembled nanoprobes serve as mass amplifiers, nanoquenchers, or nanodonors, exhibiting high FRET efficiencies (64.4–86.5%) and demonstrating excellent detection capabilities in DNA and microRNA analysis. [ABSTRACT FROM AUTHOR]

Phytochromes are photoreceptor proteins with a bilin chromophore that undergo photoconversion between two spectrally different forms, Pr and Pfr. Three domains, termed PAS, GAF, and PHY domains, constitute the N‐terminal photosensory chromophore module (PCM); the C‐terminus is often a histidine kinase module. In the Agrobacterium fabrum phytochrome Agp1, the autophosphorylation activity of the histidine kinase is high in the Pr and low in the Pfr form. Crystal structure analyses of PCMs suggest flexibility around position 308 in the Pr but not in the Pfr form. Here, we performed time‐resolved fluorescence anisotropy measurements with different Agp1 mutants, each with a single cysteine residue at various positions. The fluorophore label Atto‐488 was attached to each mutant, and time‐resolved fluorescence anisotropy was measured in the Pr and Pfr forms. Fluorescence anisotropy curves were fitted with biexponential functions. Differences in the amplitude A2 of the second component between the PCM and the full‐length variant indicate a mechanical coupling between position 362 and the histidine kinase. Pr‐to‐Pfr photoconversion induced no significant changes in the time constant t2 at any position. An intermediate t2 value at position 295, which is located in a compact environment, suggests flexibility around the nearby position 308 in Pr and in Pfr. [ABSTRACT FROM AUTHOR]

G proteins are interacting partners of G protein-coupled receptors (GPCRs) in eukaryotic cells. Upon G protein activation, the ability of the Gα subunit to exchange GDP for GTP determines the intracellular signal transduction. Although various studies have successfully shown that both Gαs and Gαi have an opposite effect on the intracellular cAMP production, with the latter being commonly described as "more active", the functional analysis of Gαs is a comparably more complicated matter. Additionally, the thorough investigation of the ubiquitously expressed variants of Gαs, Gαs(short) and Gαs(long), is still pending. Since the previous experimental evaluation of the activity and function of the Gαs isoforms is not consistent, the focus was laid on structural investigations to understand the GTPase activity. Herein, we examined recombinant human Gαs by applying an established methodological setup developed for Gαi characterization. The ability for GTP binding was evaluated with fluorescence and fluorescence anisotropy assays, whereas the intrinsic hydrolytic activity of the isoforms was determined by a GTPase assay. Among different nucleotide probes, BODIPY FL GTPγS exhibited the highest binding affinity towards the Gαs subunit. This work provides a deeper understanding of the Gαs subunit and provides novel information concerning the differences between the two protein variants. [ABSTRACT FROM AUTHOR]

In this work, we introduced a simple aggregation-induced emission enhancement (AIEE) sensor (PHCS) which can selectively detect and discriminate three environmentally and biologically imperative heavy metal ions (Cu2+, Co2+ and Hg2+) and a hazard class 1 categorized nitro-explosive picric acid (PA) in differential media. By virtue of its weak fluorescence attributes in pure organic medium owing to the synergistic operation of multiple photophysical quenching mechanisms, the molecular probe showcased highly selective 'TURN ON' fluorogenic response towards hazardous Hg2+ with a limit of detection (LOD) as low as 97 nM. Comprehensive investigation of binding mechanism throws light on the cumulative effect of probe-metal complexation induced chelation enhanced fluorescence (CHEF) effect and subsequent AIEE activation within the formed probe-metal adducts. Noteworthily, the probe (PHCS) can be readily used in real water samples for the quantitative determination of Hg2+ in a wide concentration range. In addition, the probe displayed modest colorimetric recognition performances to selectively detect and discriminate two essential heavy metal ions (Cu2+ and Co2+) with a LOD of 96 nM and 65 nM for Cu2+ and Co2+ respectively, in semi-aqueous medium. Intriguingly, based on high photoluminescence efficiency, the AIEE active nano-aggregated PHCS displayed a remarkable propensity to be used as a selective and ultra-sensitive 'TURN-OFF' fluorogenic chemosensor towards PA with LOD of 34.4 ppb in aqueous medium. Finally, we specifically shed light on the interaction of PHCS hydrosol towards PA using some unprecedented techniques, which helped uncover new photophysical insights of probe-explosive molecule interaction. We shed light on novel photophysical insights toward unique multifunctional sensory aptitude of a simple aggregation-induced emission enhancement active organic functional molecule in differential media, enabling ultra-sensitive discriminative detection of toxic heavy metal ions and explosive molecule simultaneously. [ABSTRACT FROM AUTHOR]

The study introduces a novel C3‐symmetric β‐diketone compound, BTA‐D3, and its monomeric counterpart, D, with a focus on their synthetic procedure, photophysical properties and aggregation behavior. Both compounds exhibit characteristic absorption and weak fluorescence in solution, with BTA‐D3 displaying higher absorption coefficients due to its larger number of diketone units. Density Functional Theory (DFT) calculations suggest increased co‐planarity of diketone groups in BTA‐D3. A significant finding is the Aggregation‐Induced Emission (AIE) property of BTA‐D3, as its fluorescence intensity increases dramatically when exposed to specific solvent ratios. The AIE behavior is attributed to intermolecular excitonic interaction between BTA‐D3 molecules in self‐organized aggregates. We also studied fluorescence anisotropy of BTA‐D3 and D. Despite its larger size, BTA‐D3 showed reduced anisotropy values because of efficient intramolecular energy migration among three diketone units. Furthermore, BTA‐D3 demonstrates unique polymorphism, yielding different emission colors and structures depending on the solvent used. A unique approach is presented for promoting the growth of self‐organized aggregate structures via solvent evaporation, leading to distinct fluorescence properties. This research contributes to the understanding of C3‐symmetric structural molecules and provides insights into strategies for controlling molecular alignment to achieve diverse fluorescence coloration in molecular materials. [ABSTRACT FROM AUTHOR]

The cell wall is essential for bacteria to maintain structural rigidity and withstand external osmotic pressure. In bacteria, the cell wall is composed of peptidoglycan. Lipid II is the basic unit for constructing highly cross-linked peptidoglycan scaffolds. Transglycosylase (TGase) is the initiating enzyme in peptidoglycan synthesis that catalyzes the ligation of lipid II moieties into repeating GlcNAc-MurNAc polysaccharides, followed by transpeptidation to generate cross-linked structures. In addition to the transglycosylases in the class-A penicillin-binding proteins (aPBPs), SEDS (shape, elongation, division and sporulation) proteins are also present in most bacteria and play vital roles in cell wall renewal, elongation, and division. In this review, we focus on the latest analytical methods including the use of radioactive labeling, gel electrophoresis, mass spectrometry, fluorescence labeling, probing undecaprenyl pyrophosphate, fluorescence anisotropy, ligand-binding-induced tryptophan fluorescence quenching, and surface plasmon resonance to evaluate TGase activity in cell wall formation. This review also covers the discovery of TGase inhibitors as potential antibacterial agents. We hope that this review will give readers a better understanding of the chemistry and basic research for the development of novel antibiotics. [ABSTRACT FROM AUTHOR]

We investigate the nanometrology of sub-nanometre particle sizes in industrially manufactured sodium silicate liquors at high pH using time-resolved fluorescence anisotropy. Rather than the previous approach of using a single dye label, we investigate and quantify the advantages and limitations of multiplexing two fluorescent dye labels. Rotational times of the non-binding rhodamine B and adsorbing rhodamine 6G dyes are used to independently determine the medium microviscosity and the silicate particle radius, respectively. The anisotropy measurements were performed on the range of samples prepared by diluting the stock solution of silicate to concentrations ranging between 0.2 M and 2 M of NaOH and on the stock solution at different temperatures. Additionally, it was shown that the particle size can also be measured using a single excitation wavelength when both dyes are present in the sample. The recovered average particle size has an upper limit of 7.0 ± 1.2 Å. The obtained results were further verified using small-angle X-ray scattering, with the recovered particle size equal to 6.50 ± 0.08 Å. To disclose the impact of the dye label on the measured complex size, we further investigated the adsorption state of rhodamine 6G on silica nanoparticles using molecular dynamics simulations, which showed that the size contribution is strongly impacted by the size of the nanoparticle of interest. In the case of the higher radius of curvature (less curved) of larger particles, the size contribution of the dye label is below 10%, while in the case of smaller and more curved particles, the contribution increases significantly, which also suggests that the particles of interest might not be perfectly spherical. [ABSTRACT FROM AUTHOR]

Physicochemical investigations on the inclusion of anionic polyamidoaminesuccinamic acid dendrimer, generation 5 (PAMAM-SA, G5) with positively charged hybrid vesicles (HCV), prepared using soylecithin, ion pair amphiphile (IPA), cholesterol and dihexadecyldimethylammonium bromide, were investigated by dynamic light scattering, transmission electron/atomic force microscopy (TEM/AFM), differential scanning calorimetry, fluorescence spectroscopy and surface pressure-time isotherm studies. Adsorption of dendrimer onto vesicle surface and subsequent bilayer disruption strongly depends on the bilayer composition and dendrimer concentration. Change in the zeta potential value with increasing dendrimer concentration suggests the dendrimer-vesicle interaction to be electrostatic in nature. AFM studies also confirm the adsorption of dendrimer as well as hole formation in the bilayer. Impact of the inclusion of dendrimer into the bilayer were further investigated through differential scanning calorimetry by monitoring the chain melting temperature and enthalpy of the chain melting processes. Dendrimer at low concentration does not alter bilayer integrity, while hole formations are noted at higher dendrimer concentration. Fluorescence anisotropy studies confirm the adsorption and subsequent bilayer disruption due to dendrimer inclusion. Dendrimer induced vesicle disintegration kinetics conclusively illustrate the transformation of cationic bilayer to monolayer and thereby exposing the role of IPA. In vitro cytotoxicity studies on PAMAM-SA, G5 and HCVs mixtures against human breast cancer cell line suggest that dendrimer-liposome aggregates (dendriosomes) exhibit substantial anticancer activities with insignificant side effects. It is expected that the dendriosomes may have application to host and deliver anticancer drug in the field of targeted drug delivery. [ABSTRACT FROM AUTHOR]

Alveolar septation increases gas-exchange surface area and requires coordinated cytoskeletal rearrangement in lung fibroblasts (LFs) to balance the demands of contraction and cell migration. We hypothesized that DBN (drebrin), a modulator of the actin cytoskeleton in neuronal dendrites, regulates the remodeling of the LF cytoskeleton. Using mice bearing a transgelin-Cre–targeted deletion of Dbn in pulmonary fibroblasts and pericytes, we examined alterations in alveolar septal outgrowth, LF spreading and migration, and actomyosin function. The alveolar surface area and number of alveoli were reduced, whereas alveolar ducts were enlarged, in mice bearing the dbn deletion (DBNΔ) compared with their littermates bearing only one dbn-Flox allele (control). Cultured DBNΔ LFs were deficient in their responses to substrate rigidity and migrated more slowly. Drebrin was abundant in the actin cortex and lamella, and the actin fiber orientation was less uniform in lamella of DBNΔ LFs, which limited the development of traction forces and altered focal adhesion dynamics. Actin fiber orientation is regulated by contractile NM2 (nonmuscle myosin-2) motors, which help arrange actin stress fibers into thick ventral actin stress fibers. Using fluorescence anisotropy, we observed regional intracellular differences in myosin regulatory light chain phosphorylation in control LFs that were altered by dbn deletion. Using perturbations to induce and then release stalling of NM2 on actin in LFs from both genotypes, we made predictions explaining how DBN interacts with actin and NM2. These studies provide new insight for diseases such as emphysema and pulmonary fibrosis, in which fibroblasts inappropriately respond to mechanical cues in their environment. [ABSTRACT FROM AUTHOR]

Two-dimensional polarization imaging (2D POLIM) is an experimental method where correlations between fluorescence excitation- and fluorescence emission-polarization properties are measured. One way to analyze 2D POLIM data is to apply a so-called single funnel approximation (SFA). The SFA allows for quantitative assessment of energy transfer between chromophores with identical spectra [homo-FRET (Förster resonance energy transfer)]. In this paper, we run a series of computer experiments to investigate the applicability of the analysis based on the SFA to various systems ranging from single multichromophoric systems to isotropic ensembles. By setting various scenarios of energy transfer between individual chromophores within a single object, we were able to define the borders of the practical application of SFA. It allowed us to reach a more comprehensive interpretation of the experimental data in terms of uncovering the internal arrangement of chromophores in the system and energy transfer between them. We also found that the SFA can always formally explain the data for isotropic ensembles and derived a formula connecting the energy funneling efficiency parameter and traditional fluorescence anisotropy. [ABSTRACT FROM AUTHOR]

Pancreatic islets are metabolically active micron-sized tissues responsible for controlling blood glucose through the secretion of insulin and glucagon. A loss of functional islet mass results in type 1 and 2 diabetes. Islet-on-a-chip devices are powerful microfluidic tools used to trap and study living ex vivo human and murine pancreatic islets and potentially stem cell-derived islet organoids. Devices developed over the past twenty years offer the ability to treat islets with controlled and dynamic microenvironments to mimic in vivo conditions and facilitate diabetes research. In this review, we explore the various islet-on-a-chip devices used to immobilize islets, regulate the microenvironment, and dynamically detect islet metabolism and insulin secretion. We first describe and assess the various methods used to immobilize islets including chambers, dam-walls, and hydrodynamic traps. We subsequently describe the surrounding methods used to create glucose gradients, enhance the reaggregation of dispersed islets, and control the microenvironment of stem cell-derived islet organoids. We focus on the various methods used to measure insulin secretion including capillary electrophoresis, droplet microfluidics, off-chip ELISAs, and on-chip fluorescence anisotropy immunoassays. Additionally, we delve into the various multiparametric readouts (NAD(P)H, Ca2+-activity, and O2-consumption rate) achieved primarily by adopting a microscopy-compatible optical window into the devices. By critical assessment of these advancements, we aim to inspire the development of new devices by the microfluidics community and accelerate the adoption of islet-on-a-chip devices by the wider diabetes research and clinical communities. [ABSTRACT FROM AUTHOR]

Aims This study aimed to investigate the changes of cell membrane structure and function of Issatchenkia terricola under citric acid by performing physiological analysis. Methods and results The membrane integrity, surface hydrophobicity, structure, fluidity, apoptosis, and fatty acid methyl esters composition of I. terricola WJL-G4 cells were determined by propidium iodide staining, microbial adhesion to hydrocarbon test, transmission electron microscopy analysis, fluorescence anisotropy, flow cytometry, and gas chromatography-mass, respectively. The results showed that with the increasing of citric acid concentrations, the cell vitality, membrane integrity, and fluidity of I. terricola reduced; meanwhile, apoptosis rate, membrane permeable, hydrophobicity, and ergosterol contents augmented significantly. Compared to control, the activities of Na+, K+-ATPase, and Ca2+, Mg2+-ATPase increased by 3.73-fold and 6.70-fold, respectively, when citric acid concentration increased to 20 g l−1. The cells cracked and their cytoplasm effused when the citric acid concentration reached 80 g l−1. Conclusions I. terricola could successfully adjust its membrane structure and function below 60 g l−1 of citric acid. However, for citric acid concentrations above 80 g l−1, its structure and function were dramatically changed, which might result in reduced functionality. [ABSTRACT FROM AUTHOR]

Microtubules (MTs) are key components of the eukaryotic cytoskeleton and are essential for intracellular organization, organelle trafficking and mitosis. MT tasks depend on binding and interactions with MT-associated proteins (MAPs). MT-associated protein 7 (MAP7) has the unusual ability of both MT binding and activating kinesin-1-mediated cargo transport along MTs. Additionally, the protein is reported to stabilize MTs with its 112 amino-acid long MT-binding domain (MTBD). Here we investigate the structural basis of the interaction of MAP7 MTBD with the MT lattice. Using a combination of solid and solution-state nuclear magnetic resonance (NMR) spectroscopy with electron microscopy, fluorescence anisotropy and isothermal titration calorimetry, we shed light on the binding mode of MAP7 to MTs at an atomic level. Our results show that a combination of interactions between MAP7 and MT lattice extending beyond a single tubulin dimer and including tubulin C-terminal tails contribute to formation of the MAP7-MT complex. Integrated structural data show that the MAP7 microtubule binding domain stabilizes the microtubule lattice through binding along protofilaments. Both strong and weak interactions between MAP7 and the lattice extend beyond a single tubulin dimer and include the tubulin C-terminal tails. [ABSTRACT FROM AUTHOR]

Chemokines are important immune system proteins, many of which mediate inflammation due to their function to activate and cause chemotaxis of leukocytes. An important anti‐inflammatory strategy is therefore to bind and inhibit chemokines, which leads to the need for biophysical studies of chemokines as they bind various possible partners. Because a successful anti‐chemokine drug should bind at low concentrations, techniques such as fluorescence anisotropy that can provide nanomolar signal detection are required. To allow fluorescence experiments to be carried out on chemokines, a method is described for the production of fluorescently labeled chemokines. First, a fusion‐tagged chemokine is produced in Escherichia coli, then efficient cleavage of the N‐terminal fusion partner is carried out with lab‐produced enterokinase, followed by covalent modification with a fluorophore, mediated by the lab‐produced sortase enzyme. This overall process reduces the need for expensive commercial enzymatic reagents. Finally, we utilize the product, vMIP‐fluor, in binding studies with the chemokine binding protein vCCI, which has great potential as an anti‐inflammatory therapeutic, showing a binding constant for vCCI:vMIP‐fluor of 0.37 ± 0.006 nM. We also show how a single modified chemokine homolog (vMIP‐fluor) can be used in competition assays with other chemokines and we report a Kd for vCCI:CCL17 of 14 μM. This work demonstrates an efficient method of production and fluorescent labeling of chemokines for study across a broad range of concentrations. [ABSTRACT FROM AUTHOR]

AbstractThe characterization of nucleic acid ligase activity is of great significance for the study of physiological processes. In the present work, a new method for the detection of E. coli DNA ligase is designed based on the non-covalent interaction of carbon nanotubes with dyes and ssDNA and DNA ligation reactions. When no E. coli DNA ligase is present, the ssDNA probe and dye SG I adsorb to the surface of carbon nanotubes. After centrifugation, the carbon nanotubes with DNA and dye SG I have larger molecular weights, resulting in higher fluorescence anisotropy. When E. coli DNA ligase is present, a double-stranded structure is formed, with SG I embedded in the groove of the DNA double strand. Due to the weak interaction between the DNA double-strand structure and the carbon nanotubes, after centrifugation, the supernatant does not contain the carbon nanotubes, resulting in lower fluorescence anisotropy. The feasibility of the assay was confirmed by optimizing the conditions. This method improves the accuracy of the detection with a relative error within 1%. The increase of fluorescence anisotropy in samples is small compared with in the buffer solution. Therefore, the system has good selectivity and is suitable for the characterization of enzyme activity in biological samples. Moreover, the method does not require the labeling of fluorescent molecules or a special design of nucleic acid sequences, which avoids complex probe modification. [ABSTRACT FROM AUTHOR]

The effect of the changes in buffer concentrations or any additives like surfactants in the protein samples during the analysis on the single-biomolecule diffusion is one of the hidden points in the single-molecule time-resolved measurements. In the current work, phosphoglycerate kinase (PGK) labeled with Atto-647 has been investigated on the single molecule level while it diffuses in Na2CO3 buffer at concentrations that vary from 10 mg/l up to 50 mg/l. The fluorescence lifetime of PGK labeled with Atto-647 in 50 mg/ml Na2CO3 has been measured, and it was found to be 2.7 ns. The fluorescence cross correlation of the diffused protein has also been measured, which confirms that the used samples are at a single molecule level. Time decay fluorescence anisotropy has been performed for PGK labeled with Atto-647 in different concentrations of Na2CO3, and the results confirmed that there is a clear impact on the molecular translational and rotational diffusion even with slight changes in the buffer concentration. [ABSTRACT FROM AUTHOR]

Clustered DNA damage (cluster) or a multiply damaged site, which is a region with two or more lesions within one or two helical turns, has a high mutagenic potential and causes cell death. We quantified fluorophore-labeled lesions and estimated their proximity through fluorescence anisotropy measurements depending on Förster resonance energy transfer (FRET) among the fluorophores close to each other. pUC19 plasmid DNA (2,686 base pairs) dissolved in water or 0.2 M Tris-HCl buffer at a concentration of 10 μg/μL was irradiated by several ionizing radiations with varying linear energy transfers (LET, 0.2–1890 keV/μm). Electrophilic carbonyls (aldehydes and ketones) at abasic sites (APs) produced in DNA were labeled with Alexa Fluor 488 fluorescent dyes with an O-amino functional group. Regardless of the presence or absence of the buffer, AP yields (the number of APs/base pair/Gy) tended to decrease with increasing LET, and the ratio of the AP yield (in 0.2 M Tris-HCl/in water) was less than 0.1 in the LET range of 0.2–200 keV/μm. However, in a higher LET range, the ratios were greater than 0.1. At a low dose, fluorescence anisotropy decreased with increasing LET in 0.2 M Tris-HCl, whereas, in water, this LET dependence was almost insignificant. These findings suggest that 1. the damage distribution on a DNA molecule formed by indirect effects (e.g., by hydroxyl radicals) does not depend on radiation quality and 2. greater LET radiation is more likely to produce a cluster and/or to produce a cluster with shorter distances between lesions by direct effects. This FRET-based proximity estimation of DNA lesions will contribute not only to the identification of clusters and their complexity in a whole genome, but also to the study of their repair mechanism by single-molecular level fluorescence microscopy. [ABSTRACT FROM AUTHOR]

A model for the morphology (size, shape, and crystallinity) of carbon dots (CDs) in the solid state consistent with the observed photoluminescence in solution is proposed herein. Overwhelming evidence has been collected that links the data coming from solid‐state analysis (high‐resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry (MS)) to that of solution (pulsed‐field gradient (PFG)‐NMR spectroscopy, time‐resolved fluorescence anisotropy (TRFA), and steady‐state/time‐resolved fluorescence), allowing the establishment of an overall structural model for CDs. According to this model, the so‐called carbon dots, observed under HRTEM imaging, are in fact supramolecular organized structures dynamically assembled from small to medium‐sized molecular species when the solvent is removed to give the solid form. In this way, the imaged nanoparticles (TEM/AFM) are not covalently bound entities formed during the synthetic process, but instead supramolecular entities formed by noncovalent interactions. These particles, if at all present in solution, have the form of loose associations of relatively small molecules. This study was conducted on CDs obtained from the hydrothermal carbonization (HTC) of a biomass waste (olive wet pomace). [ABSTRACT FROM AUTHOR]

Background: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). Results: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). Conclusions: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters. [ABSTRACT FROM AUTHOR]

The mini review focuses on the work carried out in our laboratory over the last two and a half decades to unravel various facets of photoinduced processes, viz. photoinduced electron transfer (PET), excited state proton transfer, energy transfer etc., using fluorescence spectroscopy and imaging. We have demonstrated that PET is manifested by formation of exciplex and that its parent spin state is authenticated by observing steady-state fluorescence in presence of magnetic field, which is quite a non-conventional set-up. It is observed that dielectric constant of the medium governs the magnetic field effect. We have synthesized and explored fluorophores which act as sensors for hydrogen bonds and protic media. Our group has studied the protonation equilibrium of Acridine, which is a good fluorophore in various confined media and observed the perturbation of this equilibrium in presence of DNA bases using steady-state and time-resolved fluorescence techniques. Excimer formation of 9-Aminoacridine Hydrochloride Hydrate (9AA), another strong fluorophore, has been investigated in several solvent matrices and photoinduced interactions of 9AA with amines and viologens are studied. Further, we have utilized fluorescence upconversion technique to segregate direct and diffusion-controlled electron transfer within picosecond-femtosecond time domain. Then attempts have been made to study DNA-ligand and protein-ligand interactions comprehensively, utilizing different techniques of fluorescence spectroscopy. Furthermore, we have synthesized fluorescent carbon dots and have made an endeavor to find their fruitful biological significance. We have also synthesized riboflavin (Rf)-gold nano-assemblies (RfS@AuNPs) by covalently attaching thiolated Rf to gold nanoparticles (AuNPs) and have observed that these RfS@AuNPs accumulate in the nucleus of cancer cells, leading to plasma membrane blebbing and binucleated apoptotic bodies, consistent with activation of apoptosis. Finally, some very recent examples as well as the future prospect of fluorescence spectroscopy have been discussed.

Time-resolved fluorescence anisotropy has been extensively used to detect changes in bimolecular rotation associated with viscosity levels within cells and other solutions. Physiological alterations of the viscosity of biological fluids have been associated with numerous pathological causes. This current work serves as proof of concept for a method to measure viscosity changes in small analyte volumes representative of biological fluids. The fluorophores used in this study were fluorescein disodium salt and Enhanced Green Fluorescent Protein (EGFP). To assess the ability of the method to accurately detect viscosity values in small volume samples, we conducted measurements with 12 µL and 100 µL samples. No statistically significant changes in determined viscosities were recorded as a function of sample volume for either fluorescent probe. The anisotropy of both fluorescence probes was measured in low viscosity standards ranging from 1.02 to 1.31 cP, representative of physiological fluid values, and showed increasing rotational correlation times in response to increasing viscosity. We also showed that smaller fluid volumes can be diluted to accommodate available cuvette volume requirements without a loss in the accuracy of detecting discrete viscosity variations. Moreover, the ability of this technique to detect subtle viscosity changes in complex fluids similar to physiological ones was assessed by using fetal bovine serum (FBS) containing samples. The presence of FBS in the analytes did not alter the viscosity specific rotational correlation time of EGFP, indicating that this probe does not interact with the tested analyte components and is able to accurately reflect sample viscosity. We also showed that freeze–thaw cycles, reflective of the temperature-dependent processes that biological samples of interest could undergo from the time of collection to analyses, did not impact the viscosity measurements' accuracy. Overall, our data highlight the feasibility of using time-resolved fluorescence anisotropy for precise viscosity measurements in biological samples. This finding is relevant as it could potentially expand the use of this technique for in vitro diagnostic systems. [ABSTRACT FROM AUTHOR]

CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes. [ABSTRACT FROM AUTHOR]

Introduction: Macrophages or T-lymphocytes triggered inflammation and, consequently, activated glial cells may contribute to neuroinflammation and neurotransmitter dysfunction in schizophrenia (SZ), while omega(n)-3 polyunsaturated fatty acids (PUFAs) can attenuate some SZ symptoms through anti-inflammatory eects. However, the correlations between macrophage/Tlymphocyte-produced cytokines and glia phenotypes, between inflammatory status and PUFAs composition, between cytokines and neurotransmitter function, and between n-3 PUFAs and neurotransmitter abnormality in SZ are unclear. Methods: Changes in T-helper (h) patterns, peripheral macrophage/glial markers, PUFAs profile, membrane fluidity, and neurotransmitter functions were evaluated in SZ patients (n = 50) and healthy controls (n = 30) using ELISA, gas chromatography, fluorescence anisotropy techniques, and HPLC, respectively. Results: Compared to the control, blood lymphocyte proliferation, the concentration of macrophage/microglia phenotype M1 markers, including cytokines IL-1β, TNF-α (Th1) and IL-6 (Th2), and astrocyte phenotype A1 marker S100β was significantly increased, while IL-17 and n-3 PUFAs contents, n-3/n-6 ratio, and membrane fluidity (FLU) were significantly decreased in SZ. Moreover, increased DA and HVA, decreased 5-HT and NE, and their metabolites appeared in SZ. Moreover, negative correlations between IL-6 and A2 marker Brain-Derived Neurotrophic Factor (BDNF) or n-3 PUFAs EPA and between IL-1β and FLU or 5HIAA, while positive correlations between EPA and 5-HIAA and between FLU and DHA were found in SZ. Discussion: These findings showed (1) no clear Th pattern, but pro-inflammatorydominant immunity occurred; (2) the pro-inflammatory pattern may result in the activated microglia M1 and astrocyte A1 phenotype; and (3) increased proinflammatory cytokines were related to decreased n-3 PUFA and decreased membrane fluidity and dysfunctional neurotransmitter systems in SZ [ABSTRACT FROM AUTHOR]

Protein‐membrane interactions play an important role in various biological phenomena, such as material transport, demyelinating diseases, and antimicrobial activity. We combined vacuum‐ultraviolet circular dichroism (VUVCD) spectroscopy with theoretical (e.g., molecular dynamics and neural networks) and polarization experimental (e.g., linear dichroism and fluorescence anisotropy) methods to characterize the membrane interaction mechanisms of three soluble proteins (or peptides). α1‐Acid glycoprotein has the drug‐binding ability, but the combination of VUVCD and neural‐network method revealed that the membrane interaction causes the extension of helix in the N‐terminal region, which reduces the binding ability. Myelin basic protein (MBP) is an essential component of the myelin sheath with a multi‐layered structure. Molecular dynamics simulations using a VUVCD‐guided system showed that MBP forms two amphiphilic and three non‐amphiphilic helices as membrane interaction sites. These multivalent interactions may allow MBP to interact with two opposing membrane leaflets, contributing to the formation of a multi‐layered myelin structure. The antimicrobial peptide magainin 2 interacts with the bacterial membrane, causing damage to its structure. VUVCD analysis revealed that the M2 peptides assemble in the membrane and turn into oligomers with a β‐strand structure. Linear dichroism and fluorescence anisotropy suggested that the oligomers are inserted into the hydrophobic core of the membrane, disrupting the bacterial membrane. Overall, our findings demonstrate that VUVCD and its combination with theoretical and polarization experimental methods pave the way for unraveling the molecular mechanisms of biological phenomena related to protein‐membrane interactions. [ABSTRACT FROM AUTHOR]

To unveil and shape the molecular connectivity in (metallo)porphyrin–carbon nanotube hybrids are of main relevance for the multiple medicinal, photoelectronic, catalytic, and photocatalytic applications of these materials. Multi-walled carbon nanotubes (MWCNTs) were modified through 1,3-dipolar cycloaddition reactions with azomethine ylides generated in situ and carrying pentafluorophenyl groups, followed by immobilization of the β-amino-tetraphenylporphyrinate Zn(II). The functionalities were confirmed via XPS and FTIR, whereas Raman spectroscopy showed disruptions on the graphitic carbon nanotube surface upon both steps. The functionalization extension, measured via TGA mass loss and corroborated via XPS, was 0.2 mmol·g−1. Photophysical studies attest to the presence of the different porphyrin–carbon nanotube connectivity in the nanohybrid. Significantly different emission spectra and fluorescence anisotropy of 0.15–0.3 were observed upon variation of excitation wavelength. Vis-NIR absorption and flash photolysis experiments showed energy/charge transfer in the photoactivated nanohybrid. Moreover, evidence was found for direct reaction of amino groups with a carbon nanotube surface in the presence of molecular dipoles such as the zwitterionic sarcosine amino acid. [ABSTRACT FROM AUTHOR]

The mixing of ILs provides an opportunity for fine tuning the physiochemical properties of ILs for various applications. However, a suitable mixture having desired properties can only be designed when the physiochemical properties of the mixtures of ILs along with their spectroscopic properties are well understood. With an aim to achieve this objective, three different mixtures with a common anion, namely, [C2C1im][C4C1im][NTf2], [C3C1pyr][C4C1pyr][NTf2], and [C3C1im][C3C1pyr][NTf2], have been investigated in the current study. Investigations have been carried out at the macroscopic level by observing the thermophysical properties, such as molar volume and thermal expansion coefficient, and at the microscopic level with time-resolved fluorescence measurements and the pulse field gradient nuclear magnetic resonance (NMR) technique. The results obtained from the thermophysical study have indicated that excess molar volume for imidazolium-based IL–IL mixtures may be linked to the free volume created by the alkyl chain of the imidazolium cation whereas for the mixture of pyrrolidinium ILs, lowering of density can give rise to free volume. Analysis of time-resolved fluorescence anisotropy data has provided clear evidence in favor of the presence of free volume in the binary mixture of ILs. NMR studies have also supported the fluorescence anisotropy data. The outcome of the present investigation reveals that the mixtures show appreciable deviation from ideal behavior and the deviation from the ideal behavior is caused due to the generation of free volume in the resultant mixture, describing these IL mixtures as quasi-ideal rather than ideal or non-ideal. [ABSTRACT FROM AUTHOR]

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and β-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to -152. In addition, we demonstrated the binding of P-BvrR to three sites within the -140 to +1 region. Two of these sites were delimited between -18 to +1 and - 99 to -76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from -140 to -122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales. [ABSTRACT FROM AUTHOR]

Single-color barcoding strategies could break the limits of spectral crosstalk in conventional intensity-based fluorescence barcodes. Fluorescence anisotropy (FA), a self-referencing quantity able to differentiate spectrally similar fluorophores, is highly attractive in designing fluorescent barcodes within a limited emission window. In this study, FA-based encoding of polystyrene (PS) microspheres was realized for the first time. The FA signals of fluorophores were stabilized inside PS microspheres owing to hampered rotational motion. Fluorescent labels were incorporated with similar emission but different structures, symmetries, and lifetimes. On the one hand, Förster Resonance Energy Transfer (FRET) including homo-FRET and hetero-FRET resulted in a decrease of steady-state FA with increasing dye loading, converting conventional intensity-based codes into FA-based codes. On the other hand, mixing dyes with different intrinsic FA values generated different FA values at the same fluorescence intensity level. Single color 5-plex FA-encoded microspheres were demonstrated and decoded on a homemade microscopic FA imaging platform in real time. The FA-encoded microspheres were successfully applied to detect the oligonucleotide of the foodborne bacterium, Bacillus cereus, without spectral crosstalk between the encoding and reporting dyes. Overall, FA-based encoding with an expanded coding capacity in the FA dimension holds great potential in multiplexed high-throughput chemical and biological analyses. [ABSTRACT FROM AUTHOR]

The unique self-assembly properties of unimer micelles are exploited for the preparation of fluorescent nanocarriers embedding hydrophobic fluorophores. Unimer micelles are constituted by a (meth)-acrylate copolymer with oligoethyleneglycol and perflurohexylethyl side chains (PEGMA90-co-FA10) in which the hydrophilic and hydrophobic comonomers are statistically distributed along the polymeric backbone. Thanks to hydrophobic interactions in water, the amphiphilic copolymer forms small nanoparticles (<10 nm), with tunable properties and functionality. An easy procedure for the encapsulation of a small hydrophobic molecule (C153 fluorophore) within unimer micelles is presented. UV–vis, fluorescence, and fluorescence anisotropy spectroscopic experimental data demonstrate that the fluorophore is effectively embedded in the nanocarriers. Moreover, the nanocarrier positively contributes to preserve the good emissive properties of the fluorophore in water. The efficacy of the dye-loaded nanocarrier as a fluorescent probe is tested in two-photon imaging of thick ex vivo porcine scleral tissue. [ABSTRACT FROM AUTHOR]

The aberrant formation of α-synuclein (Syn) aggregates, varying in size, structure and morphology, has been linked to the development of Parkinson's disease. In the early stages of Syn aggregation, large protein amyloid aggregates with sizes > 100 nm in hydrodynamic radius have been noticed. These low overall abundant large Syn aggregates are notoriously difficult to study by conventional biophysical methods. Due to the growing importance of studying the early stages of Syn aggregation, we developed a strategy to achieve this purpose, which is the study of the initial effect of the Syn protein aqueous solutions temperature rise. Therefore, the increase of the Syn aqueous solutions entropy by the initial effect of the temperature rise led to the exposure of the protein hydrophobic tyrosyl groups by not interfering with this amyloid protein aggregation. As an attempt to interpret the degree of the referred protein tyrosyl groups exposure, the classic rotameric conformations of the Nα-acetyl-L-tyrosinamide (NAYA) parent compound were used. For both NAYA and Syn, it was determined that the classic rotameric conformations involving the tyrosyl groups indeed accounted for their exposure under steady-state conditions of fluorescence, for lowest molecular species concentrations investigated at least. In this situation, Syn aggregation was observed. For the higher NAYA and Syn concentrations studied, the referred classic rotameric conformation were insufficient in such referred steady-state conditions and, for Syn, in particular, fluorescence anisotropy measurements revealed that less protein aggregation occurs along with its delay. Overall, the developed strategy by focusing on the initial effect of the temperature rise of Syn aqueous solutions in lower concentrations is suitable for informing us about the degree of this protein aggregation in solution. [ABSTRACT FROM AUTHOR]

Dopamine (DA) is an important small-molecule neurotransmitter, which is closely related to the development of many neurological diseases and has received increasing attention in the diagnosis of neurological diseases. Currently, the assays of the detection of dopamine such as electrochemical and colorimetric methods have low sensitivity, poor selectivity and susceptibility to interference, which limit the accurate quantification of dopamine. Fluorescence anisotropy immunoassay is a traditional analytical method in which the quantification is based on the change in fluorescence anisotropy values observed when fluorescence molecules are bound to a certain volume and mass of the material. Since dopamine is a small molecule with small volume and mass, we took advantage of the good photostability of the second near-infrared window (NIR-II) quantum dots (QDs) and the low spontaneous interference of the substrate, and designed a biosensor dopamine fluorescence anisotropy probe streptavidin biosensor (DFAP-SAB) based on the NIR-II QDs combined with streptavidin signal amplification to achieve rapid and separation-free detection of dopamine in human serum. The detection signal has a good linearity between 50 nM and 3000 nM with a detection limit of 11.2 nM. The application of NIR-II QDs provides the possibility of biosensor applications for complex samples. The construction of the streptavidin signal amplification device provides a new idea for small molecule detection. [ABSTRACT FROM AUTHOR]

An acidophilic heterotrophic gram-negative Acidiphilium symbioticum strain H8 is an important bacterium possessing a high capacity for heavy metal binding. As these bacteria inhabit acidic mine regions they are subjected to occasional temperature stress; thus their membrane profile is very important. Cell morphology determined by scanning and transmission electron microscopy revealed the surface and internal adaptation of the organelles. Phosphatidyl ethanolamine, phosphatidyl inositol, and phosphatidic acid are the major phospholipids affected by growth temperature. The membrane fluidity in response to temperature stress was investigated by measuring fluorescence anisotropy using 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe. Significant changes in membrane fluidity revealed the transition temperature midpoints as 11 and 38°C. To maintain the optimum fluidity increasing the percentage of cis-vaccenic acid at suboptimal and palmitic acid at the upper cardinal temperature support the evidence of membrane remodeling in different growth temperatures. The protein profile from SDS-PAGE provided information about different polypeptide bands which may be responsible for the different metal binding abilities of the strain. [ABSTRACT FROM AUTHOR]

Phototautomerism in the excited states of free-base 5, 10, 15, 20-tetrakis(4-sulfonatophenyl) porphyrin (H2TPPS4−) has been investigated combining, for the first time, advanced Electron Paramagnetic Resonance (EPR) with fluorescence and Raman spectroscopy. Triplet EPR spectroscopy, performed in protic and deuterated solvents and in the presence of photoselection, confirms the occurrence of phototautomerization and additionally suggests the formation of the cis tautomer as a minor component. The zero-field splitting parameters and triplet sublevel populations indicate that the process is slow in the triplet state. The results obtained by EPR combined with photoselection and fluorescence anisotropy have been interpreted within a model which accounts for a fast trans–trans tautomerization promoted by a spin-vibronic coupling mechanism for intersystem crossing, with an even distribution of the two trans tautomers at liquid nitrogen temperatures for H2TPPS4−. [ABSTRACT FROM AUTHOR]

Intracellular monitoring of pH and polarity is crucial for understanding cellular processes and functions. This study employed pH- and polarity-sensitive nanomaterials such as carbon dots (CDs) for the intracellular sensing of pH, polarity, and viscosity using integrated time-resolved fluorescence anisotropy (FA) imaging (TR-FAIM) and fluorescence lifetime (FLT) imaging microscopy (FLIM), thereby enabling comprehensive characterization. The functional groups on the surface of CDs exhibit sensitivity to changes in the microenvironment, leading to variations in fluorescence intensity (FI) and FLT according to pH and polarity. The FLT of CDs in aqueous solution changed gradually from 6.38 ± 0.05 ns to 8.03 ± 0.21 ns within a pH range of 2–8. Interestingly, a complex relationship of FI and FLT was observed during measurements of CDs with decreasing polarity. However, the FA and rotational correlation time (θ) increased from 0.062 ± 0.019 to 0.112 ± 0.023 and from 0.49 ± 0.03 ns to 2.01 ± 0.27 ns, respectively. This increase in FA and θ was attributed to the higher viscosity accompanying the decrease in polarity. Furthermore, CDs were found to bind to three locations in Escherichia coli: the cell wall, inner membrane, and cytoplasm, enabling intracellular characterization using FI and FA decay imaging. FLT provided insights into cytoplasmic pH (7.67 ± 0.48), which agreed with previous works, as well as the decrease in polarity in the cell wall and inner membrane. The CD aggregation was suspected in certain areas based on FA, and the θ provided information on cytoplasmic heterogeneity due to the aggregation and/or interactions with biomolecules. The combined TR-FAIM/FLIM system allowed for simultaneous monitoring of pH and polarity changes through FLIM and viscosity variations through TR-FAIM. [ABSTRACT FROM AUTHOR]

Compatible solutes can reduce the impact of salt stress on bacteria. This work describes the changes in Lactobacillus delbrueckii subsp. bulgaricus 3 (L. bulgaricus 3) cell morphology, viability, cell membrane permeability, Zeta potential, and fluidity under salt stress. The results showed that compatible solutes shortened the transition of bacterial cells to the stationary phase under salt stress from 25 to 22 h. The cell diameter decreased from 831 to 515 nm. Compatible solutes increased the number of bacteria and promoted cell growth. The number of viable bacteria increased from 4.33 × 106 to 5.71 × 108 CFU/mL. Application of compatible solutes also improved stability of the cell membrane and maintained integrity under salt stress. According to the results of fluorescence anisotropy analysis, compatible solutes increased the fluidity of the cell membrane under salt stress. These observations suggested that compatible solutes could decrease cell growth inhibition under salt stress by changing cell morphology and the cell membrane properties. [ABSTRACT FROM AUTHOR]

Determining the rate of rotation of molecules from their fluorescence anisotropy decay curves is a powerful method for studying molecular systems in biological applications. The single photon count detection systems used for this have a nonlinear dependence of the photon counting rate on the fluorescence intensity flux (photon counting loss effect), which can lead to a number of artifacts. Using metal complexes of phthalocyanines as a test sample, we have shown that such a nonlinearity can cause distortions in the determination of the fluorescence anisotropy lifetime and the asymptotic fluorescence anisotropy. We also assessed the dependence of the described phenomena on temperature and estimated the manifestations of the photon counting loss effect in the case of photobleaching of the fluorophores. [ABSTRACT FROM AUTHOR]