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Glycyrrhiza uralensis polysaccharides (GUPS) are widely applied in biomedicine and functional food due to their multiple pharmacological activities and low toxicity. Despite their widespread use, the in vivo metabolic profile of GUPS remains poorly understood. To address this gap, we developed a quantitative analysis method that involves labeling GUPS with visible fluorescein (5-DTAF) and near-infrared (NIR) fluorescein (Cy7), resulting in stable conjugates with substitution degrees of 0.81% for 5-DTAF and 0.39% for Cy7. The pharmacokinetic studies showed a biphasic elimination pattern in the blood concentration-time curve following both intravenous and oral administration, consistent with a two-compartment model. Using fluorescence quantification and NIR imaging, we observed that GUPS was distributed to various tissues, exhibiting higher concentrations particularly in liver, kidney and lung. Excretion studies indicated that feces were the major excretion pathway of GUPS after oral administration (60.98%), whereas urine was the main pathway after intravenous administration (31.16%). Notably, GUPS could be absorbed rapidly by gut (Tmax 1 ± 0.61 h) and showed a biological half-time t1/2 26.4 ± 7.72 h after oral administration. Furthermore, the Caco-2 cells uptake studies illustrated that macropinocytosis and clathrin-mediated endocytosis were participated in the transport of GUPS in intestine epithelium. This comprehensive analysis of the in vivo pharmacokinetics of GUPS not only enhances our understanding of its metabolic pathways but also establishes a foundational basis for its clinical application, optimizing its therapeutic potential and safety profile.

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In the present study, the in vivo absorption and fecal excretion of a purified fraction of polysaccharides from the fruits of Lycium barbarum L. (LBPs-4) in rats were investigated by labelling LBPs-4 with fluorescein isothiocyanate (FITC). It was found that the fluorescent labeled LBPs-4 (LBPs-4-FITC) was not detected in the plasma within 24 h following the administration of a single dose of LBPs-4-FITC (100 mg/kg of body weight) to rats, indicating that LBPs-4 was hardly absorbed in its prototype form. Instead, a smaller fragment dissociated from LBPs-4-FITC was observed in feces and was accumulated in a time-dependent manner, suggesting that LBPs-4 was excreted into the feces with a form of degradation. Meanwhile, we observed that LBPs-4-FTIC could modulate the fecal bacterial community profile via increasing the relative abundances of Bacteroides ovatus and Alistipes and promote the production of acetic acid. Furthermore, the monoculture experiment confirmed that LBPs-4 could be metabolized into smaller fragment by B. ovatus, producing acetic acid. Collectively, our study provides information on the destiny of LBPs-4 after oral administration: non-absorbed but moved to the large intestine and catabolized by gut microbiota, especially B. ovatus.

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The plant cell wall is rich in polysaccharides with high heterogeneity. Investigating the composition and structure of cell wall polysaccharides is crucial for understanding the functionalities of plant cell walls. Carbohydrate electrophoresis is a sensitive and rapid method to analyze polysaccharides qualitatively and quantitatively. The process includes digesting the polysaccharides with appropriate cleavage enzymes, labeling the reducing ends of the released oligosaccharides with a highly charged fluorophore, and separating the labeled oligosaccharides in a polyacrylamide gel via high-voltage electrophoresis. The generated fluorescence can be calculated as compared to that of oligosaccharide standards. Therefore, this is a convenient method for polysaccharide characterization that can be performed in most laboratories. Here, we introduce the detailed operational steps and precautions, which are helpful for researchers to quickly obtain the structural information of polysaccharides.

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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.

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Fluorescence labeling with N-(1-naphthyl)ethylenediamine is highly effective for quantifying oxidized reducing end groups (REGs) in cellulosic materials. When combined with size exclusion chromatography in DMAc/LiCl, along with fluorescence / multiple-angle laser light scattering / refractive index detection, a detailed profile of C1-oxidized REGs relative to the molecular weight distribution of the cellulosic material can be obtained. In this work, the derivatization process was extensively optimized, to be carried out heterogeneously in the solvent N-methyl-2-pyrrolidone. Furthermore, we show that to achieve high selectivity for carboxyl groups at the C1 position, keto and aldehyde groups need to be selectively reduced (e.g., by NaBH4), and carboxyl groups other than at C1 need to be blocked (e.g., by methylation with (trimethylsilyl)diazomethane) prior to fluorescence labeling of carboxyl groups at C1 position. Finally, we demonstrate the practical value of the analytical method by measuring the content of the C1-oxidized REGs in cellulose samples after chemical (by Pinnick oxidation) or enzymatic (by treatment with C1-oxidizing LPMO enzymes) oxidation of various pulp samples.

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(1) Background: Polygonatum cyrtonema is a medicinal plant, and its polysaccharides are used for immunomodulation and the treatment of hyperglycemia. Investigation of the tissue distribution and pharmacokinetics of P. cyrtonema polysaccharide can further elucidate its pharmacological mechanisms. (2) Methods: A fluorescence-labeling approach using rhodamine B (RhB) as a fluorescent molecular probe was used for the quantitative assessment of the polysaccharide from dried P. cyrtonema (DPC1) samples, and the pharmacokinetics and tissue distribution of DPC1 were evaluated in mice after intraperitoneal or oral administration. (3) Results: DPC1 was successfully labeled with RhB, showing degrees of fluorescence labeling at 0.453% and 0.568% as determined by the ultraviolet and enzyme marker methods, respectively. DPC1-RhB was rapidly absorbed into the bloodstream after oral and intraperitoneal administration. Pharmacokinetic characteristics showed that oral administration and intraperitoneal administration were consistent with the features of a two-compartment model. (4) Conclusion: After administration, DPC1-RhB was primarily distributed in the tissues of the heart, spleen, and lung, indicating that the drug has a targeted effect on these tissues. Overall, the findings provide a comprehensive reference for the in vivo distribution of DPC1, together with a foundation for further elucidation of its pharmacological mechanisms and the development and application of DPC1 formulations.

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RNAs play important roles in regulating biological growth and development. Advancements in RNA-imaging techniques are expanding our understanding of their function. Several common RNA-labeling methods in plants have pros and cons. Simultaneously, plants' spontaneously fluorescent substances interfere with the effectiveness of RNA bioimaging. New technologies need to be introduced into plant RNA luminescence. Aggregation-induced emission luminogens (AIEgens), due to their luminescent properties, tunable molecular size, high fluorescence intensity, good photostability, and low cell toxicity, have been widely applied in the animal and medical fields. The application of this technology in plants is still at an early stage. The development of AIEgens provides more options for RNA labeling. Click chemistry provides ideas for modifying AIEgens into RNA molecules. The CRISPR/Cas13a-mediated targeting system provides a guarantee of precise RNA modification. The liquid-liquid phase separation in plant cells creates conditions for the enrichment and luminescence of AIEgens. The only thing that needs to be looked for is a specific enzyme that uses AIEgens as a substrate and modifies AIEgens onto target RNA via a click chemical reaction. With the development and progress of artificial intelligence and synthetic biology, it may soon be possible to artificially synthesize or discover such an enzyme.

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Palladium-catalyzed coupling reactions are versatile and powerful tools for the construction of carbon-carbon bonds in organic synthesis. Although these reactions have favorable features that proceed selectively in mild reaction conditions using aqueous organic solvents, no attention has been given to their application in the field of biomedical analysis. Therefore, we focused on these reactions and evaluated the scope and limitations of their analytical performance. In this review, we describe the pros and cons and future trends of fluorescence derivatization of pharmaceuticals and biomolecules based on palladium-catalyzed coupling reactions such as Suzuki-Miyaura coupling, Mizoroki-Heck coupling, and Sonogashira coupling reactions for HPLC analysis.

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Objective To observe the effectiveness of fluorescence labeling-based assay bundle intervention in the prevention and control of multidrug-resistant organism(MDRO)infection.Methods Patients who were detected MDRO in a hospital from January to December 2022 were selected as the research subjects.MDRO monitoring data and implementation status of prevention and control measures were collected.Fluorescence labeling assay was adopted to monitor the cleaning and disinfection effectiveness of the surrounding object surface of the bed units.Based on the bundled prevention and control measures as well as management mode of the pre-intervention group,the post-intervention group implemented enhanced rectification measures for the problems found by the pre-interven-tion group.Changes in relevant indicators between January-June 2022(before intervention)and July-December 2022(after intervention)were compared.Results There were 136 MDRO-infected patients in the pre-intervention group,208 MDRO strains were detected and 10 healthcare-associated infection(HAI)occurred.There were 128 MDRO-infected patients in the post-intervention group,198 MDRO strains were detected and 9 HAI occurred.Af-ter intervention,the total detection rates of methicillin-resistant Staphylococcus aureus(MRSA),carbapenem-re-sistant Acinetobacter baumannii(CRAB),and total MDRO from patients decreased significantly compared to before intervention(all P＜0.05).After intervention,the detection rates of MRSA,carbapenem-resistant Pseudomonas aeruginosa(CRPA),CRAB,and total MDRO from the surrounding object surface were all lower than those before intervention(all P＜0.05).The detection rate of MDRO from surrounding object surface before intervention was 34.52％,which showed a decreased trend after intervention(P＜0.05).The clearance rate of fluorescent labeled markers before intervention was 41.84％,which showed an upward trend after implementing intervention measures(from July to December),and increased to 85.00％at the end of intervention(November-December).The comp-liance rates of issuing isolation medical orders,placing isolation labels,using medical supplies exclusively,and cor-rectly handling medical waste after intervention have all increased compared to before intervention(all P＜0.05).Conclusion Adopting fluorescence labeling-based assay bundle intervention can effectively improve the effectiveness of MDRO infection prevention and control.

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4-Hydroxyphenylpyruvate dioxygenase (HPPD) plays a crucial role in the synthesis of nutrients needed to maintain optimal plant growth. Its level is closely linked to the extent of abiotic stress experienced by plants. Moreover, it is also the target of commercial herbicides. Therefore, labeling of HPPD in plants not only enables visualization of its tissue distribution and cellular uptake, it also facilitates assessment of abiotic stress of plants and provides information needed for the development of effective environmentally friendly herbicides. In this study, we created a method for fluorescence labeling of HPPD that avoids interference with the normal growth of plants. In this strategy, a perylene-linked dibenzyl-cyclooctyne undergoes strain-promoted azide-alkyne cycloaddition with an azide-containing HPPD ligand. The activation-based labeling process results in a significant emission enhancement caused by the change in the fluorescent forms from an excimer to a monomer. Notably, this activated bioorthogonal strategy is applicable to visualizing HPPD in Arabidopsis thaliana, and assessing its response to multiple abiotic stresses. Also, it can be employed to monitor in vivo levels and locations of HPPD in crops. Consequently, the labeling strategy will be a significant tool in investigations of HPPD-related abiotic stress mechanisms, discovering novel herbicides, and uncovering unknown biological functions.

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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. Supplementary Information: The online version contains supplementary material available at 10.1007/s43657-023-00094-1.

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Poor visualization of polyps can limit colorectal cancer screening. Fluorescent antibodies to mucin5AC (MUC5AC), a glycoprotein upregulated in adenomas and colorectal cancer, could improve screening colonoscopy polyp detection rate. Adenomatous polyposis coli flox mice with a Cdx2-Cre transgene (CPC-APC) develop colonic polyps that contain both dysplastic and malignant tissue. Mice received MUC5AC-IR800 or IRdye800 as a control IV and were sacrificed after 48 h for near-infrared imaging of their colons. A polyp-to-background ratio (PBR) was calculated for each polyp by dividing the mean fluorescence intensity of the polyp by the mean fluorescence intensity of the background tissue. The mean 25 µg PBR was 1.70 (±0.56); the mean 50 µg PBR was 2.64 (±0.97); the mean 100 µg PBR was 3.32 (±1.33); and the mean 150 µg PBR was 3.38 (±0.87). The mean PBR of the dye-only control was 2.22 (±1.02), significantly less than the 150 µg arm (p-value 0.008). The present study demonstrates the ability of fluorescent anti-MUC5AC antibodies to specifically target and label colonic polyps containing high-grade dysplasia and intramucosal adenocarcinoma in CPC-APC mice. This technology can potentially improve the detection rate and decrease the miss rate of advanced colonic neoplasia and early cancer at colonoscopy.

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Processing of newly synthesized polypeptides is essential for protein homeostasis and cell viability. In bacteria and eukaryotic organelles, all proteins are synthesized with formylmethionine at their N-terminus. As the nascent peptide emerges from the ribosome during translation, the formyl group is removed by peptide deformylase (PDF), an enzyme that belongs to the family of ribosome-associated protein biogenesis factors (RPBs). Because PDF is essential in bacteria but not in humans (except for the PDF homolog acting in mitochondria), the bacterial enzyme is a promising antimicrobial drug target. While much of the mechanistic work on PDF was carried out using model peptides in solution, understanding the mechanism of PDF in cells and developing effective PDF inhibitors requires experiments with its native cellular substrates, i.e., ribosome-nascent chain complexes. Here, we describe protocols to purify PDF from Escherichia coli and to test its deformylation activity on the ribosome in multiple-turnover and single-round kinetic regimes as well as in binding assays. These protocols can be used to test PDF inhibitors, to study the peptide specificity of PDF and its interplay with other RPBs, as well as to compare the activity and specificity of bacterial and mitochondrial PDFs.

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Tissue clearing refers to laboratory methods that make tissue transparent by chemical means. This approach allows the labeling, visualization, and analysis of specific targets without cutting the tissue into sections, thereby maintaining three-dimensional architecture. More than two dozen tissue-clearing methods have been developed by different research teams to date. While tissue clearing has been successfully applied in several studies concerning basic science or diseases, little is known about the utilization of tissue clearing for neurotoxicity evaluation. In this study, several tissue-clearing methods were combined with Fluoro-Jade C (FJ-C), a standard marker of neurodegeneration. The results suggest that some but not all tissue-clearing media are compatible with the FJ-C fluorophore. By utilizing a neurotoxicity animal model, the results further suggest that FJ-C labeling can be combined with tissue clearing for neurotoxicity assessments. This approach has the potential to be expanded further by combining multicolor labeling of molecular targets involved in the development and/or mechanisms of neurotoxicity and neurodegeneration.

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Super-resolution fluorescence microscopy techniques are powerful tools to investigate polymer systems. In this review, we address how these techniques have been applied to hydrogel nano- and microparticles, so-called nano- or microgels. We outline which research questions on microgels could be addressed and what new insights could be achieved. Studies of the morphology, shape, and deformation of microgels; their internal compartmentalization; the cross-linker distribution and polarity inside them; and their dynamics and diffusion are summarized. In particular, the abilities to super-resolve structures in three dimensions have boosted the research field and have also allowed researchers to obtain impressive 3D images of deformed microgels. Accessing information beyond 3D localization, such as spectral and lifetime properties and correlative imaging or the combination of data with other methods, shines new light onto polymer systems and helps us understand their complexity in detail. Such future trends and developments are also addressed.

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Dibutyl phthalate (DBP) is an organic pollutant frequently detected in soil, and is a reproductive poison that harms animals both before and after birth and has mutagenic, teratogenic, and carcinogenic effects. DBP removal from farmland has been the subject of extensive research in recent years. Efficient DBP degrading bacterial strains were screened in the laboratory. GFP (Green fluorescent protein) labeled degradation strain GFP-DNB-S1 was analyzed for its activity and dynamics. Using sodium alginate (SA) and nano-hydroxyapatite (n-HAP) as carrier materials and CaCl2 as a cross-linking agent, the immobilized microbial agent n-HAP/SA + DNB-S1 was prepared by embedding cross-linking immobilization technology to study the remediation effect of DBP contaminated soil. The best formation effect of immobilized materials (n-HAP/SA) was found when the SA to n-HAP ratio was 3:2. When compared to single SA immobilized bacteria, n-HAP/SA immobilized bacteria improved the surface roughness and porosity of the microspheres. After 70 days, LED light revealed that the immobilized bacteria's GFP green fluorescent protein expression was stable. At 70 days, the initial DBP concentration of 500 mg ∙ L-1 degraded at a rate of 69.9%. The degrading bacteria had no effect on DBP degradation before and after being labeled with GFP. The n-HAP/SA immobilized bacteria offered a better living environment for microorganisms due to their rougher surface and a greater number of pores. This protected the microorganisms and increased the efficiency of DBP degradation. When the concentration of DBP in contaminated soil was set to 20 mg ∙ kg-1 and the n-HAP/SA + DNB-S1 immobilized bacterial agent was applied to the soil, the rate of DBP degradation was determined to be 93.34%. The degradation process followed First-order degradation kinetics, which improved the physical and chemical properties of the soil as well as its fertility.

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Cytoplasmic dynein, the largest and most intricate cytoskeletal motor protein, powers the movement of numerous intracellular cargos toward the minus ends of microtubules (MT). Despite its essential roles in eukaryotic cells, dynein's molecular mechanism, the regulatory functions of its subunits and accessory proteins, and the consequences of human disease mutations on dynein force generation remain largely unclear. Recent work combining mutagenesis, single-molecule fluorescence, and optical tweezers-based force measurement have provided valuable insights into how dynein's multiple AAA+ ATPase domains regulate dynein's attachment to MTs. Here, we describe detailed protocols for the measurements of the force-dependent dynein-MT detachment rates. We provide updated and optimized protocols for the expression and purification of a tail-truncated single-headed Saccharomyces cerevisiae dynein, for polarity-marked MT polymerization, and for the non-covalent attachment of MTs to cover glass surfaces for the measurement of dynein-MT detachment forces.

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Rationale: Hepatic lymphatics are essential for liver homeostasis and immune function. However, the 3D structure and spatial distribution of hepatic lymphatic vessels (LVs) need to be confirmed. Moreover, the molecular information of hepatic lymphatic endothelial cells (LyECs) needs to be further studied. The bottleneck is the lack of specific markers or labeling methods for hepatic lymphatic endothelial cells (LyECs) Methods: Here, we proposed a method for the spatiotemporal sequential injection of antibodies (STSI-Ab) to selectively label hepatic LyECs in vivo. In addition, we also developed an efficient hepatic LyEC sorting method and performed deep transcriptome sequencing on hepatic LyECs. Results: The STSI-Ab method achieved selective labeling of the mouse hepatic lymphatic network. Three-dimensional fluorescence imaging results of the STSI-Ab mouse liver lobe clearly showed that hepatic LVs entangled with the portal vein but were not present in the central vein. The imaging data inspired a novel hepatic lobule structure model with an added set of LVs in the portal area. Furthermore, deep transcriptome sequencing of isolated hepatic LyECs and Masson's trichrome staining results suggested that hepatic LyECs might be an important source of collagen fibers deposited in the portal area during the process of liver fibrosis and bile duct ligation (BDL). Conclusions: We proposed an STSI-Ab method for selectively labeling hepatic LVs, distinguishing the hepatic LVs from other vessels, and mapping their 3D structure. This study opens an avenue for understanding hepatic lymphatic structure and it will be very beneficial to the study of hepatic LyEC functions.

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The processes of genome expression, regulation, and repair require direct interactions between proteins and DNA at specific sites located at and near single-stranded-double-stranded DNA (ssDNA-dsDNA) junctions. Here, we review the application of recently developed spectroscopic methods and analyses that combine linear absorbance and circular dichroism spectroscopy with nonlinear 2D fluorescence spectroscopy to study the local conformations and conformational disorder of the sugar-phosphate backbones of ssDNA-dsDNA fork constructs that have been internally labeled with exciton-coupled cyanine (iCy3)2 dimer probes. With the application of these methods, the (iCy3)2 dimer can serve as a reliable probe of the mean local conformations and conformational distributions of the sugar-phosphate backbones of dsDNA at various critical positions. The results of our studies suggest a possible structural framework for understanding the roles of DNA breathing in driving the processes of protein-DNA complex assembly and function.

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Expansion microscopy enables super-resolved visualization of specimen without the need of highly sophisticated and expensive optical instruments. Instead, the method is executed with conventional chemicals and lab equipment. Imaging of bacteria is performed using standard fluorescence microscopy. This chapter describes a protocol for the expansion microscopy of Bacillus subtilis expressing DivIVA-GFP. In addition, the cell wall was labeled by wheat germ agglutinin. Here, we place emphasis on the challenges of selecting the protein and organism of interest.

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