Fluorescence Experiments Research Articles

Lamin A/C Downregulation Alters Prostate Cancer Cellular Phenotypes

Nuclear lamina proteins are associated with the structural stability of cells and the anchoring and localization of heterochromatin. The downregulation of lamin A/C has been shown to contribute to cellular deformity, genome instability, and affect the ability of cells to differentiate. Therefore, evaluating lamins in prostate cancer progression may uncover important mechanisms for coordinated regulation of cellular processes, thereby highlighting potential vulnerabilities that may be targeted therapeutically to alter the clinical course of the disease. Previous multiplex fluorescent immunolabeling experiments showed a substantial fraction of prostate cancers displaying dramatically diminished lamin A/C expression in the cancer cells compared to the benign prostate epithelial (luminal and basal) cell populations. In this study, we investigate how downregulation of lamin A/C directly affects cellular characteristics associated with metastasis. By knocking down lamin A/C expression (~50%) with siRNAs targeting lamin A/C, we tested how the modulation of lamin A/C directly affected cell proliferation, clonogenic survival, and cell invasion in three prostate cancer cell lines (PC3, DU145, and CWR22Rv1). Compared to a scrambled siRNA control, all three cell lines showed no significant change in proliferation and clonogenic survival when lamin A/C was downregulated. While PC3 and DU145 showed no significant change in invasion potential, lamin A/C knockdown in CWR22Rv1 resulted in a decreased invasion potential of 22.8% compared to controls. We are conducting further experiments to fully elucidate the effects of lamin A/C downregulation on these metastasis‐relevant cellular properties. Overall, these findings will help to further understand the significance of alterations in the nuclear lamina protein's involvement in metastatic progression, and to further validate lamin dysregulation measurements as a potential biomarker to predict clinical outcomes in prostate cancer.

A Research on the Role and Mechanism of N-Methyl-D-Aspartate Receptors in the Effects of Silver Nanoparticles on the Electrical Excitability of Hippocampal Neuronal Networks

The purpose of this paper is to explore the role and mechanism of N-Methyl-D-Aspartate (NMDA) receptors in the effects of silver nanoparticles (SNPs) on the electrical excitability of hippocampal neuronal networks. First, the cytotoxicity of different concentrations of SNPs was evaluated and screened by MTT experiment, then the Voltage Threshold Measurement Method (VTMM) was employed to study the effects of SNPs on the electrical excitability of hippocampal neuronal networks under non-cytotoxic (5 μM) and cytotoxic (100 μM) concentrations after different action times. The role of NMDA receptors in the effects of SNPs on the electrical excitability of hippocampal neuronal networks was investigated through the NMDA receptor antagonist MK-801. Then, the effects of SNPs on the number of NMDA receptors and the Ca2+ content in hippocampal neurons were further investigated, and the relationship between these changes and neuronal networks electrical excitability was discussed. The results of voltage threshold (VTh) test showed that non-cytotoxic 5 μM SNPs has an excitatory effect on hippocampal neuronal networks, while the effect of cytotoxic 100 μM SNPs gradually changed from excitatory to inhibitory with the extension of action time. It was found that SNPs could increase the electrical excitability of neuronal networks by activating NMDA receptors through the experiments with MK-801 antagonists. Moreover, the fluorescent staining experiments showed that the activation of NMDA receptors by SNPs can lead to an increase in the intracellular Ca2+ content, and then trigger a negative feedback regulation mechanism of neurons between the number of NMDA receptors and intracellular Ca2+ content. The high Ca2+ content in neurons can also decrease neurons’ cell viability, which in turn leads to changes in the electrical excitability of the neuronal networks.

Turn-on Fluorescent Biosensors for Imaging Hypoxia-like Conditions in Living Cells.

We present the synthesis, photophysical properties, and biological application of nontoxic 3-azo-conjugated BODIPY dyes as masked fluorescent biosensors of hypoxia-like conditions. The synthetic methodology is based on an operationally simple N=N bond-forming protocol, followed by a Suzuki coupling, that allows for a direct access to simple and underexplored 3-azo-substituted BODIPY. These dyes can turn on their emission properties under both chemical and biological reductive conditions, including bacterial and human azoreductases, which trigger the azo bond cleavage, leading to fluorescent 3-amino-BODIPY. We have also developed a practical enzymatic protocol, using an immobilized bacterial azoreductase that allows for the evaluation of these azo-based probes and can be used as a model for the less accessible and expensive human reductase NQO1. Quantum mechanical calculations uncover the restructuration of the topography of the S1 potential energy surface following the reduction of the azo moiety and rationalize the fluorescent quenching event through the mapping of an unprecedented pathway. Fluorescent microscopy experiments show that these azos can be used to visualize hypoxia-like conditions within living cells.

Flexible terahertz metamaterial biosensor for label-free sensing of serum tumor marker modified on a non-metal area.

Terahertz (THz) metamaterials for rapid label-free sensing show application potential for the detection of cancer biomarkers. A novel flexible THz metamaterial biosensor based on a low refraction index parylene-C substrate is proposed. The biomarkers are modified on non-metal areas by a three-step modification method that simplifies the modification steps and improves the modified effectivity. Simulation results for non-metal modification illustrate that a bulk refractive index sensitivity of 325 GHz/RIU is achieved, which is larger than that obtained for the traditional metal modification (147 GHz/RIU). Meanwhile, several fluorescence experiments proved the uniform modification effect and selective adsorption capacity of the non-metal modification method. The concentration of the carcinoembryonic antigen (CEA) biomarkers for breast cancer patients tested using this THz biosensor is found to be consistent with results obtained from traditional clinical tests. The limit of detection reaches 2.97 ng/mL. These findings demonstrate that the flexible THz metamaterial biosensor can be extensively used for the rapid detection of cancer biomarkers in the future.

Aggregation-Induced Emission with Alkynylcoumarin Dinuclear Gold(I) Complexes: Photophysical, Dynamic Light Scattering, and Time-Dependent Density Functional Theory Studies.

Aggregation-induced emission (AIE) has gained a remarkable amount of interest in the past 20 years, but the majority of the studies are based on organic structures. Herein, three dinuclear gold(I) complexes, with the general formula [PPh2XPPh2-Au2-Coum2], where the Au(I) atom is linked to three different diphosphanes [PPh2XPPh2; DPPM for X = CH2 (1.1), DPPP for X = (CH2)3 (1.2), and DPPA for X = C≡C (1.3)] and the propynyloxycoumarin precursor (1, 4-methyl-substituted coumarin), have been synthesized. The compounds present AIE characteristics, AIEgens, with high luminescence quantum yields in the solid state when they are compared to dilute solutions. Photophysical studies (steady-state and time-resolved fluorescence) were obtained, with AIE being observed with the three gold(I) complexes in acetonitrile/water mixtures. This was further corroborated with dynamic light scattering measurements. Time-dependent density functional theory (TDDFT) electronic calculations show that the compounds have different syn and anti conformations (relative to the coumarin core) with 1.1 syn and 1.2 and 1.3 both anti. From time-resolved fluorescence experiments, the augment in the contribution of the longer decay component is found to be associated with the emission of the aggregate (AIE effect) and its nature (involving a dimer) rationalized from TDDFT electronic calculations.

Thalidomide-based Pt(IV) prodrugs designed to exert synergistic effect of immunomodulation and chemotherapy

Combination of immune- and chemo-therapy has become a new trend in cancer treatment. Food and Drug Administration (FDA)-approved immune-modulatory agent, thalidomide, can modulate the related proteins of upstream signaling pathway of programmed cell death-ligand 1 (PD-L1), including nuclear transcription factor κB (NF-κB), hypoxia inducible factor-1α (HIF-1α), epidermal growth factor receptor (EGFR), and signal transducer and activator of transcription 3 (STAT3), all acting as key antitumor target proteins. In this work, we conjugated thalidomide with oxidized cisplatin to construct multi-functional Pt(IV) prodrugs, named thaliplatins 4–6, to investigate the anti-tumor effect of immuno- and chemo-therapy. Among them, thaliplatin 6 exerted remarkable cytotoxicity against the tested cancer cell lines, showing 15–26 and 9–20 times higher IC50 values than those of single cisplatin or the combination of cisplatin + thalidomide, respectively. Moreover, thaliplatin 6 could rapidly accumulated into cells, markedly triggered DNA damage, and induced cell S phase arrest and apoptosis, as well as inhibited cell migration and invasion in breast carcinoma cell line (MCF-7). Fluorescent confocal and western blotting experiments proved that 6 significantly regulated NF-κB, EGFR, HIF-1α and phosphor-signal transducer and activator of transcription 3 (p-STAT3), and simultaneously inhibited PD-L1 expression to interrupt programmed cell death 1 (PD-1)/PD-L1 signaling pathway, suggesting a synergistic action of cisplatin and thalidomide. Most strikingly, in vivo tests indicated that 6 effectively decreased tumor growth with no observable systemic toxicity, being superior to the anticancer efficacy of cisplatin.

Investigating the binding mechanism of topiramate with bovine serum albumin using spectroscopic and computational methods.

Various spectroscopic techniques involving fluorescence spectroscopy, circular dichroism (CD), and computational approaches were used to elucidate the molecular aspects of interaction between the antiepileptic drug topiramate and the multifunctional transport protein bovine serum albumin (BSA) under physiological conditions. Topiramate quenched BSA fluorescence in a static quenching mode, according to the Stern-Volmer quenching constant (Ksv ) data derived from fluorescence spectroscopy for the topiramate-BSA complex. The binding constant was also used to calculate the binding affinity for the topiramate-BSA interaction. Fluorescence and circular dichroism experiments demonstrate that the protein's tertiary structure is affected by the microenvironmental alterations generated by topiramate binding to BSA. To establish the exact binding site, interacting residues, and interaction forces involved in the binding of topiramate to BSA, molecular modeling and simulation approaches were used. According to the Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) calculations, the average binding energy between topiramate and BSA is -421.05 kJ/mol. Topiramate was discovered to have substantial interactions with BSA, changing the structural dynamic and Gibbs free energy landscape patterns.

Characteristics and structure of a soy protein isolate-lutein nanocomplex produced via high-pressure homogenization.

In recent years, nanocarriers for transporting active substances have attracted attention. This study was to explore the soy protein isolate (SPI) after high-pressure homogenization (HPH) (0, 30, 60, 90 and 120 MPa) as potential lutein carriers. The load amount (LA) and encapsulation efficiency (EE) of the SPI-lutein nanocomplexes at a homogenization pressure of 60 MPa were the highest (2.32 mg mL-1 and 92.85%, respectively), and the average particle size and ζ-potential of the SPI-lutein nanocomplexes were 192.1nm and -30.06 mV, respectively. The DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydroxyl-antioxidant activities of the complex increased from 12.4% and 23.3% to 52.7% and 61.07%, respectively, after the protein was treated with HPH. The surface hydrophobicity of the SPI and the SPI-lutein nanocomplexes increased with increasing homogenization pressure treatment. Fourier transform-infrared spectrophotometry analyses suggested that the homogenization treatments resulted in partial unfolding of the protein molecules, and the addition of lutein can also lead to the change of protein secondary structure. The fluorescence emission of SPI was quenched by lutein through the static quenching mechanism. Fluorescence experiments revealed that SPI and lutein had the strongest binding ability through hydrophobic interaction at a homogenization pressure of 60 MPa. After HPH, the combination of SPI and lutein was beneficial, and the stability of lutein also improved after the combination. This study is conducive to expanding the application of soybean protein in the food industry. © 2022 Society of Chemical Industry.

Integrated Analysis of the ceRNA Network and M-7474 Function in Testosterone-Mediated Fat Deposition in Pigs.

Castration can significantly enhance fat deposition in pigs, and the molecular mechanism of fat deposition caused by castration and its influence on fat deposition in different parts of pigs remain unclear. RNA-seq was performed on adipose tissue from different parts of castrated and intact Yorkshire pigs. Different ceRNA networks were constructed for different fat parts. GO and KEGG pathway annotations suggested that testosterone elevates cell migration and affects differentiation and apoptosis in back fat, while it predisposes animals to glycolipid metabolism disorders and increases the expression of inflammatory cytokines in abdominal fat. The interaction between M-7474, novel_miR_243 and SGK1 was verified by dual fluorescence experiments. This ceRNA relationship has also been demonstrated in porcine preadipocytes. Overexpression of M-7474 significantly inhibited the differentiation of preadipocytes compared to the control group. When 100 nM testosterone was added during preadipocyte differentiation, the expression of M-7474 was increased, and preadipocyte differentiation was significantly inhibited. Testosterone can affect preadipocyte differentiation by upregulating the expression of M-7474, sponging novel-miR-243, and regulating the expression of genes such as SGK1. At the same time, HSD11B1 and SLC2A4 may also be regulated by the corresponding lncRNA and miRNA, which ultimately affects glucose uptake by adipocytes and leads to obesity.

Metal-organic framework sensors based on triazole carboxylic acid ligands for ion sensing and dye adsorption

Three novel metal-organic frameworks (MOFs) [Cd(L)(Ata)(H2O)]·H2O(1)、[Cd2(L)2(Bda)(NO3−)H2O]·2H2O(2) and [Cd2(L)2(Tpa)0.5(NO3−)(H2O)3](3) (Ata ​= ​2-aminoterephthalic acid; Bda ​= ​Succinic acid; Tpa ​= ​Terephthalic acid) were synthesized by reactions of cadmium(II) salt with organic ligands of 3, 5-bis-(triazol-1-yl)-benzoic acid (HL) and 2-aminoterephthalic acid, succinic acid, and terephthalic acid. The crystal structure test revealed that they are three different complexes. Among them, complex 1 is connected by auxiliary ligands to form a two-dimensional layered structure, and complexes 2 and 3 are connected by auxiliary ligands to form a high-dimensional three-dimensional framework structure. Solid-state fluorescence experiments showed that complexes 1 and 2 have excellent optical properties: Complex 1 can selectively detect Cu2+ (LOD: 1.97 ​μM) and MnO4− (LOD: 1.12 ​μM), and complex 2 can selectively detect Fe3+ (LOD: 1.99 ​μM) and MnO4− (LOD: 4.70 ​μM). Amino acid and antibiotic sensing experiments showed that: complex 1 can detect arginine by fluorescence enhancement effect (LOD: 5.40 ​μM), and tetracycline hydrochloride can be detected by quenching effect (LOD: 0.43 ​μM); complex 2 can be detected tryptophan (LOD: 5.72 ​μM) and tetracycline hydrochloride (LOD: 0.63 ​μM).

Modeling and Characterization of Exciplexes in Photoredox CO2 Reduction: Insights from Quantum Chemistry and Fluorescence Spectroscopy.

Interactions between excited-state arenes and amines can lead to the formation of structures with a distinct emission behavior. These excited-state complexes or exciplexes can reduce the ability of the arene to participate in other reactions, such as CO2 reduction, or increase the likelihood of degradation via Birch reduction. Exciplex geometries are necessary to understand photophysical behavior and probe degradation pathways but are challenging to calculate. We establish a detailed computational protocol for calculation, verification, and characterization of exciplexes. Using fluorescence spectroscopy, we first demonstrate the formation of exciplexes between excited-state oligo-(p-phenylene) (OPP), shown to successfully carry out CO2 reduction, and triethylamine. Time-dependent density functional theory is employed to optimize the geometries of these exciplexes, which are validated by comparing both emission energies and their solvatochromism with the experiment. Excited-state energy decomposition analysis confirms the predominant role played by charge transfer interactions in the red shift of emissions relative to the isolated excited-state OPP*. We find that although the exciplex emission frequency depends strongly on solvent dielectric, the extent of charge separation in an exciplex does not. Our results also suggest that the formation of solvent-separated ionic radical states upon complete electron transfer competes with exciplex formation in higher-dielectric solvents, thereby leading to reduced exciplex emission intensities in fluorescence experiments.

A novel Cd (II) coordination polymer of highly sensitive sensing for antibiotics in aqueous medium

A novel Cd(II) coordination polymer (CP) {[Cd(H2DOBPDC2-)(bpd)]·H2O}n (1) has been synthesized by a rigid polycarboxylic acid with hydroxyl groups (H4DOBPDC = 4,4′- dihydroxy-[1,1′-biphenyl-3,3′-dicarboxylic acid]). Structure analysis displayed that CP 1 shows a 3D supramolecular structure through hydrogen bonding. In addition, fluorescence experiments confirmed 1 can sensitively and selectively detect NFT in aqueous medium with limit of detection of 3.67 × 10-6 M. Meanwhile, the detection mechanism of 1 toward NFT was also investigated by the experiments of PXRD, UV–vis spectrum and fluorescent lifetime and Density Functional Theory (DFT) calculation in detail.

Enhancement of the Water Affinity of Histidine by Zinc and Copper Ions.

Histidine (His) is widely involved in the structure and function of biomolecules. Transition-metal ions, such as Zn2+ and Cu2+, widely exist in biological environments, and they are crucial to many life-sustaining physiological processes. Herein, by employing density function calculations, we theoretically show that the water affinity of His can be enhanced by the strong cation–π interaction between His and Zn2+ and Cu2+. Further, the solubility of His is experimentally demonstrated to be greatly enhanced in ZnCl2 and CuCl2 solutions. The existence of cation–π interaction is demonstrated by fluorescence, ultraviolet (UV) spectroscopy and nuclear magnetic resonance (NMR) experiments. These findings are of great importance for the bioavailability of aromatic drugs and provide new insight for understanding the physiological functions of transition metal ions.

Constructing bifunctional Co3O4@Ni3S2 as pair of electrooxidations catalysts with superior photoelectrocatalytic efficiency for water purification

Finding a bifunctional heterostructure catalyst that can act as a pair of electrooxidations to decompose refractory organic dyes in wastewater is an important issue. Herein, we succeeded in preparing a Co3O4@Ni3S2 heterostructure by the two-step hydrothermal method as promising bifunctional catalysts that function simultaneously as the anode and cathode. Remarkably, the optimized bifunctional Co3O4@Ni3S2-3.0 electrodes exhibit a degradation rate of ~ 95% and acceptable stability for practical usage, which can be attributed to its low Tafel slope, large electrochemically active surface area, and low charge transfer resistance. Free radical capture experiments using auxiliary probe molecular fluorescence experiments suggest that the Z-scheme heterostructure of Co3O4@Ni3S2-3.0 is proposed to promote charge separation and produce the main active species. The rational design of this work--a new strategy for establishing a highly efficient bifunctional pair of electrooxidation heterostructure catalysts of Co3O4@Ni3S2-3.0 with a Z-scheme band structure—will lead to other catalysts designs.

ESIPT and PET-based easy-to-synthesize unsymmetrical ligand in the reversible fluorimetric sensing of Al3+ and relay sensing of inorganic and biological phosphates

We have developed an unsymmetrical Schiff-base ligand H3L which selectively senses Al3+ over all other metal ions in ethanol–water mixture (1:1). There is dramatic increase in the intensity of fluorescence on addition of Al3+ to the solution of ligand in EtOH/H2O mixture. NMR studies, HRMS results and fluorescence experiments have been conducted to confirm the binding mode of different metals with H3L. The detection limit for aluminum ion is 72.6 × 10−9 M. The L-Al3+ complex has been subsequently used to detect inorganic and organic phosphates. An “On-Off-On” relay recognition of Al3+ ion and different phosphate and pyrophosphate ions, ATP and ADP has been achieved. The probe has shown good reversibility behavior based on which truth table and logic gate was designed. Lifetime and quantum yield values have been measured in an effort to investigate the sensing ability of the ligand. Cost effective real-time detection of Al3+ has been demonstrated by filter paper strips.

A Series of Photosensitizes for Fe3+ in Aqueous Solution and Cells With Colorimetric and Fluorescent Channels: Synthesis and Performance.

Ferrum (Fe) is a widely existing metal element and nearly the most important trace element in living species, including human beings. The design of chemosensors for Fe ions faces issues related to the d-d transition of Fe(II) and Fe(III) ions, which makes them efficient electron trappers and energy quenchers. Most fluorescent dyes cannot afford such d-d quenching, showing emission turn off effect towards both Fe(II) and Fe(III) ions with poor selectivity. As a consequence, the development for Fe with emission turn on effect and good selectivity shall be continued and updated. In this work, three rhodamine-derived chemosensors modified by different lengths of alkyl chains having electron-donating N and O atoms were synthesized and explored for the selective optical sensing of Fe(III). These chemosensors showed colorimetric and fluorescent emission turn on sensing for Fe(III), showing two sensing channels. These chemosensors showed good selectivity, which was assigned to the sieving effect of alkyl chains with electron-donating N and O atoms. The N atom was found to be more effective in associating with Fe(III), compared to the O atom. Their fluorescent cell imaging experiment was carried out to confirm the possibility of being used for cell imaging.

Mechanistic Insights into the Photoisomerization of N,N'-Disubstituted Indigos.

N,N′‐disubstituted indigos are photoswitchable molecules that have recently caught the attention due to their addressability by red‐light. When alkyl and aryl groups are utilized as the N‐substituents, the thermal half‐lives of Z isomers can be tuned independently while maintaining the advantageous red‐shifted absorption spectra. To utilize these molecules in real‐world applications, it is of immense importance to understand how their molecular structures as well as the environment influence their switching properties. To this end, we probed their photoisomerization mechanism by carrying out photophysical and computational studies in solvents of different polarities. The fluorescence and transient absorption experiments suggest for more polar excited and transition states, which explains the bathochromic shifts of absorption spectra and shorter thermal half‐lives. On the other hand, the quantum chemical calculations reveal that in contrast to N‐carbonyl groups, N‐alkyl and N‐aryl substituents are not strongly conjugated with the indigo chromophore and can thus serve as a tool for tuning the thermal stability of Z isomers. Both approaches are combined to provide in‐depth understandings of how indigos undergo photoswitching as well as how they are influenced by N‐substituent and the chemical surroundings. These mechanistic insights will serve as guiding principles for designing molecules eyeing broader applications.

Phenylalanine and indole effects on the pathogenicity of human lysozyme amorphous aggregates

Protein aggregation can affect the stability and function of proteins, and may lead to developing diseases, but reports on the in vivo effect of aggregates are scarce.In the current study, the effect of phenylalanine (Phe) and indole presence was first investigated on the structure and stability of human lysozyme (HLZ) and its aggregation under in vitro condition. Tm measurements, circular dichroism and spectrofluorimetric spectra, as well as and transmission electron microscopy (TEM) were performed in this stage. In the next step, pathogenicity of HLZ amorphous aggregates formed in presence or absence of the additives was investigated in vivo, by subcutaneous injection to adult male Wistar rats. Resulting inflamed tissues were studied by hematoxylin and eosin (HE), Congo red and Sudan black staining. Serum levels of liver enzymes (Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST)), specific inflammatory cytokines (Tumor Necrosis Factor Alpha (TNF-α) and Interleukin 6 (IL-6)) as well as glucose, cholesterol, and triglyceride levels were measured.Amorphous aggregates of HLZ caused inflammation and affected the number of fat cells, macrophages, cytokines, liver enzymes and glucose. Indole, that increases amorphous aggregates amount as shown with CD, fluorescence, and TEM experiments, leads into more severe inflammation. In presence of Phe, (which stabilizes HLZ structure) a markedly milder inflammatory state is observed in histological results and no increase could be detected in the inflammation-related parameters.In conclusion, amorphous aggregates of HLZ may be pathogenic in vivo, and presence of anti-aggregation compounds (such as Phe) can be effective in diminishing their deleterious manifestations.

A “traffic jam” of (+)-catechin caused by hyperglycemia — The interaction between (+)-catechin and human serum albumin (HSA) in high glucose environment

(+)-catechin is an active natural substance for hyperglycemia. However, HSA may be affected by high blood glucose, which alters HSA in the secondary structure to affect the combination of (+)-catechin and HSA, resulting in a “traffic jam” of (+)-catechin in the body. In this study, the effect of glucose on the binding between (+)-catechin and HSA was studied by multiple analysis techniques. It was found that glucose could not alter the static quenching of (+)-catechin-HSA. Electrostatic force played an important role through thermodynamics parameters. The binding constants were 4.956 × 105 mol⋅L-1, ΔH° = –33.6 kJ⋅mol−1, and ΔS° = 99.68 J⋅mol−1⋅K−1 in the glucose-free environment and the binding constants were 4.602 × 104 mol⋅L-1, ΔH° = −20.4 kJ⋅mol−1 and ΔS° = 57.7 J⋅mol−1⋅K−1 in the glucose environment at 298 K. We found that the binding sites of (+)-catechin in two environments were site II through the probe test. 3D fluorescence and CD experiments indicated the addition of (+)-catechin changed the secondary structure of HSA. Besides, molecular docking manifested that glucose could affect the combine of HSA and (+)-catechin. (+)-catechin had weaker binding to HSA in the glucose environment, which was different from the combination of (+)-catechin and HSA alone. The dosage of (+)-catechin for diabetic patients should be reduced because the concentration of free (+)-catechin would increase in the body. This study had a certain guiding significance for the medication of diabetic patients.

Analysis tools for single-monomer measurements of self-assembly processes

Protein assembly plays an important role throughout all phyla of life, both physiologically and pathologically. In particular, aggregation and polymerization of proteins are key-strategies that regulate cellular function. In recent years, methods to experimentally study the assembly process on a single-molecule level have been developed. This progress concomitantly has triggered the question of how to analyze this type of single-filament data adequately and what experimental conditions are necessary to allow a meaningful interpretation of the analysis. Here, we developed two analysis methods for single-filament data: the visitation analysis and the average-rate analysis. We benchmarked and compared both approaches with the classic dwell-time-analysis frequently used to study microscopic association and dissociation rates. In particular, we tested the limitations of each analysis method along the lines of the signal-to-noise ratio, the sampling rate, and the labeling efficiency and bleaching rate of the fluorescent dyes used in single-molecule fluorescence experiments. Finally, we applied our newly developed methods to study the monomer assembly of actin at the single-molecule-level in the presence of the class II nucleator Cappuccino and the WH2 repeats of Spire. For Cappuccino, our data indicated fast elongation circumventing a nucleation phase whereas, for Spire, we found that the four WH2 motifs are not sufficient to promote de novo nucleation of actin.