In situ visualization of the cellular uptake and sub-cellular distribution of mussel oligosaccharides

Unlike chemosynthetic drugs designed for specific molecular and disease targets,active small-molecule natural products typically have a wide range of bioactivities and multiple targets,necessitating extensive screening and development.To address this issue,we propose a strategy for the direct in situ microdynamic examination of potential drug candidates to rapidly identify their effects and mechanisms of action.As a proof-of-concept,we investigated the behavior of mussel oligosaccharide(MOS-1)by tracking the subcellular dynamics of fluorescently labeled MOS-1 in cultured cells.We recorded the entire dynamic process of the localization of fluorescein isothiocyanate(FITC)-MOS-1 to the lysosomes and visualized the distribution of the drug within the cell.Remarkably,lysosomes containing FITC-MOS-1 actively recruited lipid droplets,leading to fusion events and increased cellular lipid consumption.These drug behaviors confirmed MOS-1 is a candidate for the treatment of lipid-related diseases.Furthermore,in a high-fat HepG2 cell model and in high-fat diet-fed apolipoprotein E(ApoE)^(-/-)mice,MOS-1 significantly promoted triglyceride degradation,reduced lipid droplet accumulation,lowered serum triglyceride levels,and mitigated liver damage and steatosis.Overall,our work supports the prioritization of in situ visual monitoring of drug location and distribution in subcellular compartments during the drug development phase,as this methodology contributes to the rapid identification of drug indications.Collectively,this methodology is significant for the screening and development of selective small-molecule drugs,and is expected to expedite the identification of candidate molecules with medicinal effects.

Preparation, Characterization and Pharmacokinetics of Fluorescence Labeled Propylene Glycol Alginate Sodium Sulfate

A rapid and sensitive fluorescence labeling method was developed and validated for the microanalysis of a sulfated polysaccharide drug,namely propylene glycol alginate sodium sulfate(PSS), in rat plasma. Fluorescein isothiocyanate(FITC) was selected to label PSS, and 1, 6-diaminohexane was used to link PSS and FITC in order to prepare FITC-labeled PSS(F-PSS) through a reductive amination reaction. F-PSS was identified by UV-Vis, FT-IR and 1H-NMR spectrum. The cell stability and cytotoxicity of F-PSS were tested in Madin-Darby canine kidney(MDCK) cells. The results indicated that the labeling efficiency of F-PSS was 0.522% ± 0.0248% and the absolute bioavailability was 8.39%. F-PSS was stable in MDCK cells without obvious cytotoxicity. The method was sensitive and reliable; it showed a good linearity, precision, recovery and stability. The FITC labeling method can be applied to investigating the absorption and metabolism of PSS and other polysaccharides in biological samples.

Visualization of integrin molecules by fluorescence imaging and techniques

Integrin molecules are transmembraneαβheterodimers involved in cell adhesion,trafficking,and signaling.Upon activation,integrins undergo dynamic conformational changes that regulate their affinity to ligands.The physiological functions and activation mechanisms of integrins have been heavily discussed in previous studies and reviews,but the fluorescence imaging techniques-which are powerful tools for biological studies-have not.Here we review the fluorescence labeling methods,imaging techniques,as well as Förster resonance energy transfer assays used to study integrin expression,localization,activation,and functions.

The LB Films of Dansyl Chloride Labeled Octadecylamine and Its Fluorescence Lifetime

Octadecylamine was derivatized with dansyl chloride (5-dimethylaminonaphthalene-1-sulfonyl chloride) In order to simplify and understand the LB films of fluorescent probe labeling proteins. its monolayer and multilayers in the absence and presence of stearic acid were deposited by LB technique. Fluorescence spectra and lifetimes of the fluorescent products were studied to elucidate the microenvironment of molecules in the LB films.

Kinetics of Coil-to-Globule Transition of DansyI-Labeled Poly（N-isopropyl- acrylamide） Chains in Aqueous Solution

The coil-to-globule transition of thermally sensitive linear poly（N-isopropylacrylamide） （PNIPAM） labeled with dansyl group is induced by 1.54 μm laser pulses （widths10 ns）. The dansyl group is used to follow the transition kinetics because its fluorescence intensity is very sensitive to its micro-environment. As the molar ratio of NIPAM monomer to dansyl group increases from 110 to 300, the effect of covalently attached dansyl fluorophores on the transition decreases. In agreement with our previous study in which we used 8-anilino- l-naphthalensulfonic acid ammonium salt free in water as a fluorescent probe, the current study reveals that the transition has two distinct stages with two characteristic times, namely, Tfast≈0.1 ms, which can be attributed to the nucleation and formation of some ＂pearls＂ （locally contracting segments） on the chain, and tslow≈0.5 ms, which is related to the merging and coarsening of the ＂pearls＂.Tfast is independent of the PNIPAM chain length over a wide range （Mw=2.8× 10^6-4.2 × 10^7 g/mol）. On the other hand, Tslow only slightly increases with the chain length.

Intrahepatic transplantation of hepatic oval cells for fulminant hepatic failure in rats

AIM： To evaluate the effect of intrahepatic transplantation of hepatic oval cells （HOC） on fulminant hepatic failure （FHF） in rats.METHODS： HOC obtained from rats were labeled with green fluocescent protein （GFP） or 5, 6- carboxyfluorescein diacetate succinmidyl ester （CFDASE）. Cell fluorescence was observed under fluorescent microscope at 6, 24, 48 and 72 h after labeling. CFDA- SE labeled HOC （5 × 10^6 cells each rat） were injected into livers of rats with FHF induced by D-galactosamine. Serum albumin （ALB）, alanine aminotransferase （ALT）, aspartate aminotransferase （AST） and total bilirubin （TBil） levels were measured at different time points. Liver function of rats was examined on days 3, 7, 14 and 21 after HOC transplantation.RESULTS： The positive rate of GFP and CFDA-SE labeled HOC was 10% and 90%, respectively, with no significant change in cell viabilities. The survival rate was higher in HOC transplantation group than in control group, especially 48 （9/15 vs 6/15） and 72 h （9/15 vs 4/15） after HOC transplantation. The serum ALT, AST and TBil levels were decreased while the serum AIb level was increased after HOC transplantation. Fluorescence became faded and diffused in liver tissues, suggesting that proliferation and differentiation occur in transplanted HOC.CONCLUSION： CFDA-SE is superior to GFP in labeling HOC, although fluorescence intensity is decreased progressively with cell division. HOC transplantation can improve the liver function and increase the survival rate of recipients.

Effect of Tb/Mg doping on composition and physical properties of hydroxyapatite nanoparticles for gene vector application

Transfection efficiency of hydroxyapatite nanoparticles(HAnps)is relative to the particle size,morphology,surface charge,surface modifier and so on.This study prepared HAnps with doped Tb/Mg by hydrothermal synthesis method(HTSM)and investigated the effects of different Tb/Mg contents on the morphology,particle size,surface charge,composition and cellular endocytosis of HAnps.The results showed that Mg-HAnps possessed better dispersion ability than Tb-HAnps.With increasing doping content of Tb/Mg-HAnps,the granularity of Tb-HAnps increased,while that of Mg-HAnps declined.Both particle size and zeta potential of Mg-HAnps were lower than those of Tb-HAnps.7.5%Mg-doping HAnps presented relatively uniform slender rod morphology with average size of30nm,while10%Mg-doping HAnps were prone to agglomeration.Moreover,Mg-HAnps-GFP(green fluorescent protein)endocytosed by MG63cells was dotted in the perinuclear region,while Tb-HAnps were more likely to aggregate.In conclusion,as gene vectors,Mg-HAnps showed enhanced properties compared to Tb-HAnps.

Preparation and Performance of Fluorescein Isothiocyanate⁃Labeled Fluorescent Starch and Polyvinyl Alcohol for Warp Sizing

In order to solve the problems of low accuracy and poor variety adaptability of the current measurement method of the permeability and coating property of sizing paste in textile industry, taking starch and polyvinyl alcohol(PVA) as the representatives of the most commonly used textile sizes, various concentrations of fluorescent molecules fluorescein isothiocyanate(FITC) were used to label starch and PVA to prepare fluorescein(F)-starch and F-PVA fluorescent sizes with different degrees of labeling(DLs) for the first time, respectively. Then the starch and PVA derivatives were employed to size pure cotton warp yarns. After preparing the sections of the sized yarns, the permeability and coating percentage of starch and PVA paste to the yarns were calculated by using a fluorescence microscope and the Photoshop software, respectively. The results demonstrate that F-starch and F-PVA with appropriate DL of 0.791% and 0.161%, respectively, exhibit good fluorescence property and similar sizing performance to the sizing performance of unlabeled starch and PVA. It is considered that fluorescence labeling of sizing agents with fluorescein units can provide an innovative way for the accurate determination of the permeability and coating property of sizing paste to warp yarns.

The osteogenetic rate in alveolar bone remodeling induced by distraction osteogenesis of the periodontal ligament

Objective: To observe osteogenetic rate of alveolar bone on the tension side in orthodontic tooth movement through distraction osteogenesis of the periodental ligament quantificationally. Methods: The experiment was carried in 6 dogs. The left side of jaws of each one was set as test or control side, and the other side was control or test side. On the control side, the first premorlar was moved by traditional method on the test side. A self-made distraction device was used on the test side. The newly formed alveolar bone on the tension side of moved tooth was labeled by serial tetracycline fluorochrome. Sections were observed by fluorescence microscope and pictured. Newly formed bone was measured by computer image analysis. Results: The quantity of newly formed bone was significantly different between the two methods. Newly formed bone in rapid tooth movement by distraction osteogenesis of the periodental ligament was more than that in traditional method. Conclusion: The distraction through periodental ligament could induce more rapid bone formation and excite higher osteogenetic activity than traditional method.

Virtual Fluorescence Translation for Biological Tissue by Conditional Generative Adversarial Network

Fluorescence labeling and imaging provide an opportunity to observe the structure of biological tissues,playing a crucial role in the field of histopathology.However,when labeling and imaging biological tissues,there are still some challenges,e.g.,time-consuming tissue preparation steps,expensive reagents,and signal bias due to photobleaching.To overcome these limitations,we present a deep-learning-based method for fluorescence translation of tissue sections,which is achieved by conditional generative adversarial network(cGAN).Experimental results from mouse kidney tissues demonstrate that the proposed method can predict the other types of fluorescence images from one raw fluorescence image,and implement the virtual multi-label fluorescent staining by merging the generated different fluorescence images as well.Moreover,this proposed method can also effectively reduce the time-consuming and laborious preparation in imaging processes,and further saves the cost and time.

A Convenient Fluorescent-labeled Assay for in vitro Measurement of DNA Mismatch Repair Activity

Objective The assay of DNA mismatch repair （MMR） activity can be used as a biomarker for environmental condition detection and human disease diagnosis. Radioactive 32p-endlabeled DNA containing mismatch is extensively used as the substrate for MMR activity analyses. The aim of the present study is to develop a simple non-radioactive, but equally specific and sensitive method for the MMR activity assay. Methods A fluorescent label was chosen to replace the radioactive isotope label. Sensitive evaluation of the fluorescent label was carried out for the first time, and then the fluorescent label was compared with the isotope label in the MMR activity and DNA binding assays. Result LOD （limit of detection） of the fluorescent label was about 0.1 fmol and the relative signal strength displayed a pretty good linear relationship. Moreover, the fluorescent label method has equivalent sensitivity and performance as compared with the classical radioactive method in experiments. Conclusion In light of the sensitivity, reproducibility, safety, rapidity and long lifespan of the fluorescent label, this improved method can be applied to evaluation of biologic and toxic effects of environmental pollutants on man and other forms of life.

RGDC Peptide Modified Quantum Dots Labelling and Imaging of Tumor Cells

The labelling and imaging of tumor cells were investigated via arginine-glycine-aspartic acidcysteine（RGDC） peptide-labelled quantum dots（QDs）. The results show that RGDC modified QDs can label SMMC-7721 tumor cells and adhere to cellular membrane. In constrast, the unmodified QDs are mainly dispersed around the cell. We also found that the RGDC-QDs can penetrate into the cell at 2 h of incubation. After 6 h of incubation, RGDC-QDs can accumulate in a unique intracellular region.

Construction of bicistronic green fluorescent protein labeled pSELECT GFPzeo human bone morphogenetic protein 2 eukaryotic expression vector

Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase

利用定量单病毒示踪技术探究SARS-CoV-2与细胞膜融合动态过程

Viral envelope fusion with the host plasma membrane(PM)for genome release is a hallmark step in the life cycle of many enveloped viruses.This process is regulated by a complex network of biomolecules on the PM,but robust tools to precisely elucidate the dynamic mechanisms of virus-PM fusion events are still lacking.Here,we developed a quantitative single-virus tracking approach based on highly efficient dual-color labelling of viruses and batch trajectory analysis to achieve the spatiotemporal quantification of fusion events.This approach allows us to comprehensively analyze the membrane fusion mechanism utilized by pseudotyped severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)at the singlevirus level and precisely elucidate how the relevant biomolecules synergistically regulate the fusion process.Our results revealed that SARS-CoV-2 may promote the formation of supersaturated clusters of cholesterol to facilitate the initiation of the membrane fusion process and accelerate the viral genome release.

Effects of Fluorescent Pair on the Kinetics of DNA Strand Displacement Reaction

Fluorescent labels are widely used in the characterizations of DNA-based reaction network operations.We systematically studied the effects of commonly used fluorescent pairs on thermal stabilities of signal-substrate duplex and the strand displacement kinetics.It is demonstrated that the modifications of duplex with fluorescent pairs stabilize DNA duplex by up to 3.5°C,and the kinetics of DNA strand displacement circuit is also evidently slowed down.These results highlight the importance of fluorescent pairs towards the kinetic modulation in designing nucleic acid probes and complex DNA dynamic circuits.

Synthesis of photolabile dUTP analogues and their enzymatic incorporation for DNA labeling

Two new photolabile nucleotide analogues with furan-fused deoxyuridine were synthesized through Sonogashira coupling.Their enzymatic incorporation into DNA was evaluated with two DNA polymerases(Taq and Deep vent exo-)by polymerase chain reaction(PCR).Deep vent exo-recognized both nucleotides as substrates for primer extension,while Taq was much less proficient.Light irradiation of PCR products released the amino and carboxyl moieties of DNA.Further labeling with fluorescein isothiocyanate for a long DNA construct with F-dUnTP incorporation was successfully achieved.

An automated fluorescent single strand conformation polymorphism technique for high throughput mutation screening

Objective To develop a high throughput mutational detection method by mutiple fluorescence-labeled polymerase chain reaction(PCR)products.Methods A total of 27 known mutations including 22 substitutions,3 insertions(1,2 and 7 bp)and 2 deletions(1 and 2 bp)in the hepatocyte nuclear factor(HNF)-4α,glucokinase and HNF-1α genes were tested.During nested PCR,amplified fragments were labeled with three fluorescent dyes.PCR products were visualized with an ABI-377 fluorescence sequencer using 5% glycerol or 10% sucrose in nondenaturing gel conditions.Results Twenty-five of 27 variants(93%)could be detected by combining 5% glycerol and 10% sucrosegel matrix conditions.Twenty-two of 27(82%)and 18 of 27(67%)variants were identified using 5%glycerol and 10% sucrose conditions,respectively.Conclusion This fluorescence-based PCR single strand conformation polymorphism technique represents a simple,non-hazardous,time-saving and sensitive method for high throughput mutation detection.

Determination of genetic relationships between evergreen azalea cultivars in China using AFLP markers

Evergreen azaleas are among the most important ornamental shrubs in China.Today,there are probably over 300 cultivars preserved in different nurseries,but with little information available on the cultivar itself or relationships between cultivars.Amplified fragment length polymorphism(AFLP) markers were employed to determine the genetic relationships between evergreen azalea cultivars in China.One hundred and thirty genotypes collected from gardens and nurseries,including cultivars classified in the groups East,West,Hairy,and Summer,unknown cultivars,and close species,were analyzed using three primer pairs.A total of 408 polymorphic fragments were generated by AFLP reactions with an average of 136 fragments per primer pair.The average values of expected heterozygosity and Shannon's information index were 0.3395 and 0.5153,respectively.Genetic similarities were generated based on Dice coefficients,used to construct a neighbor joining tree,and bootstrapped for 100 replicates in Treecon V1.3b.Principal coordinate analysis(PCO) was performed based on Dice distances using NTSYS-pc software.The AFLP technique was useful for analyzing genetic diversity in evergreen azaleas.Cluster analysis revealed that cultivars in the West and Summer groups were quite distinct from other groups in the four-group classification system and that the East and Hairy groups should be redefined.

Blue light-induced rare-earth free phosphors for the highly sensitive and selective imaging of latent fingerprints based on enhanced hydrophobic interaction

The demand for in-situ detection of latent fingerprints(LFPs)in ways of high sensitivity,high selectivity,high contrast,low cost and user-friendly is still urgent.To overcome this challenge,a moisture-stable,red-emitting fluoride phosphor K_(3)AlF_(6):Mn^(4+)(KAF:Mn^(4+))with an organic hydrophobic skin was prepared.The phosphor has a uniform and superfine morphology with excellent luminescence properties.More importantly,this non-ultraviolet(UV)or non-near infrared(NIR)induced phosphor was proved to be an ideal fluorescent label for LFP imaging,which is both friendly for touch DNA analysis and compatible to forensic light sources.The well-defined ridge details with little background interference on various surfaces were presented by the oleic acid(OA)modified KAF:Mn^(4+)(KAF:Mn^(4+)-OA)phosphor in few seconds using the powder dusting method.To confirm the high selectivity of KAF:Mn^(4+)-OA for LFP imaging,an efficient quantitative evaluation method is proposed with the aid of ImageJ&Origin software.Due to the superiority of the Mn^(4+)-doped fluoride for the rapid imaging of LFPs in terms of lowcost,high compatibility and good availability,it is expected to be a promising candidate for forensic science as well as fluorescence imaging in other fields instead of rare earth luminescent materials.

Shaping Small Bioactive Molecules to Untangle Their Biological Function： A Focus on Fluorescent Plant Hormones

Modern biology overlaps with chemistry in explaining the structure and function of all cellular processes at the molecular level. Plant hormone research is perfectly located at the interface between these two disciplines, taking advantage of synthetic and computational chemistry as a tool to decipher the complex biological mechanisms regulating the action of plant hormones. These small signaling molecules regulate a wide range of developmental processes, adapting plant growth to ever changing environmental conditions. The synthesis of small bioactive molecules mimicking the activity of endogenous hormones allows us to unveil many molec- ular features of their functioning, giving rise to a new field, plant chemical biology. In this framework, fluores- cence labeling of plant hormones is emerging as a successful strategy to track the fate of these challenging molecules inside living organisms. Thanks to the increasing availability of new fluorescent probes as well as advanced and innovative imaging technologies, we are now in a position to investigate many of the dynamic mechanisms through which plant hormones exert their action. Such a deep and detailed comprehension is mandatory for the development of new green technologies for practical applications. In this review, we sum- marize the results obtained so far concerning the fluorescent labeling of plant hormones, highlighting the basic steps leading to the design and synthesis of these compelling molecular tools and their applications.