Frontiers and development in live-cell super-resolution fluorescence microscopy / 生物医学工程学杂志

This paper reviews the research progress on live-cell super-resolution fluorescence microscopy, discusses the current research status and hotspots in this field, and summarizes the technological application of super-resolution fluorescence microscopy for live-cell imaging. To date, this field has gained progress in numerous aspects. Specifically, the structured illumination microscopy, stimulated emission depletion microscopy, and the recently introduced minimal photon fluxes microscopy are the current research hotspots. According to the current progress in this field, future development trend is likely to be largely driven by artificial intelligence as well as advances in fluorescent probes and relevant labelling methods.

Genetic code expansion and its application in characterization and imaging of proteins / 生物工程学报

Genetic code expansion (GCE) allows the incorporation of unnatural amino acids into proteins via using stop codons. GCE may achieve site-specific labeling of proteins in combination with the click reaction. Compared with other labeling tools such as fluorescent proteins and tagged antibodies, the compound molecules used in protein labeling by GCE technology are smaller, and therefore, may less interfere the conformational structure of proteins. In addition, through click reaction, GCE allows a 1:1 stoichiometric ratio of the target protein molecule and the fluorescent dye, and the protein can be quantified based on the fluorescence intensity. Thus, GCE technology has great advantages in the researches that require the exposition of living cells under high laser power for longer time, for example, in the context of single molecule tracing and super-resolution microscopic imaging. Meanwhile, this technology lays the foundation for improving the accuracy of positioning and molecule counting in the imaging process of living cells. This review summarized the GCE technology and its recent applications in functionally characterizing, labeling and imaging of proteins.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Fluorescent antibiotics for real-time tracking of pathogenic bacteria / 药物分析学报

The harm of pathogenic bacteria to humans has promoted extensive research on physiological processes of pathogens, such as the mechanism of bacterial infection, antibiotic mode of action, and bacterial antimicrobial resistance. Most of these processes can be better investigated by timely tracking of fluorophore-derived antibiotics in living cells. In this paper, we will review the recent development of fluorescent antibiotics featuring the conjugation with various fluorophores, and focus on their applica-tions in fluorescent imaging and real-time detection for various physiological processes of bacteria in vivo.

Rapid screening of potential analgesic ingredients from Draconis Resina by live cell immobilized chromatography coupled with HPLC-DAD-TOF-MS / 中草药

Objective: To rapidly screen the potential analgesic ingredients from Draconis Resina by live cell immobilized chromatography coupled with HRMS. Methods: An HPLC-DAD-ESI-TOF-MS technique was used to rapidly identify the main chemical constituents from Draconis Resina. Based on the bio-specific affinity adsorption of bioactive compounds with receptors or channels on cells, the potential bioactive components in Draconis Resina could be selectively bound to the target cells-mice dorsal root neurons cells, then the chemical constituents with cell target affinity were identified by LC-HRMS. Results: A total of 21 compounds with various structures were tentatively identified and characterized by HPLC-DAD-ESI-TOF-MS, and among them, 10 potentially analgesic active ingredients in Draconis Resina extract combined with dorsal root neurons cells were successfully detected and identified, including two stilbene, two homoisoflavones, one homoisoflavone, two dihydrochalcone, and three flavonoid oligomers. Conclusion: Live cell immobilized chromatography coupled with LC-DAD-HRMS analysis could provide a rapid and efficient tool for finding the potential bioactive components in Draconis Resina for the next pharmacology studies, which provide the reference for exploring of effective materials basis in Chinese medicines.

Selection and identification of aptamers for mesenchymal stem cells from different species / 军事医学

Objective To screen an ssDNA aptamer for rabbit mesenchymal stem cells (MSCs) and to identify the ability of the aptamer to recognize MSCs of a variety of species origin.Methods MSCs were isolated from the thigh bone of immature rabbits and identified by induced osteogenic and adipogenic differentiation,respectively.Aptamers were screened by cell SELEX (systematic evolution of ligands by exponential enrichment) technique targeting to isolated MSCs.Enrichment of the 5th pool was evaluated through binding assay of FAM modified pool to MSCs by confocal microscopy.The enriched 5th pool was then cloned into pGE-T vector and the cloned sequences were determined randomly.The candidates were chosen based on primary sequence conservation and predicted secondary structure by RNA structure and MEME online analysis.Flow cytometry analysis was used to identify the aptamers binding to MSCs of rabbit, rat, and human origin.Results The isolated MSCs had the potential of osteogenic differentiation and adipogenic differentiation under certain conditions.Aptamer 5-1-12 from 5th enriched pool was characterized as MSCs recognizing aptamer binding to MSCs of rabbit, rat and human origin.Conclusion Aptamer 5-1-12 that recognizes MSCs of different species origin is obtained through live cell-SELEX.

Recent Advances in Fluorescent Probes for Imaging of Superoxide Anion Radical / 分析化学

Superoxide anion radical ( O·-2 ) is the first generated reactive oxygen species ( ROS) and plays essential function in life processes. Normal level of O·-2 as important signaling molecular can regulate redox equilibrium, cellular proliferation and differentiation. However, abnormal level of O·-2 is closely associated with diseases, such as cancer, neurodegenerative diseases and diabetes. Hence it is significant to uncover diseases mechanism by exploring dynamic regulation of O·-2 . Considering the advantages of fluorescence imaging method, the key factor is to develop O·-2 probes with highly selective and sensitive properties for revealing the molecular mechanism of diseases. Recently, with the development of fluorescence microscopy, many fluorescent probes have been constructed and applied for imaging analysis of O·-2 . In this review, we mainly summarized the progress of O·-2 fluorescent probes according to the different probe structure and prospected the development directions of O·-2 probes.

Mechanisms of cholic acid for reducing damage to human live cell lines L-O2 induced by amanita toxic peptides / 中华实用儿科临床杂志

Objective To explore the mechanisms of different cholic acid for reducing damage to human liver cells lines L-O2 induced by amanita toxic peptides (amataxins).Methods According to different concentrations of amataxins,the experiment was conducted with different dosages in 5 groups:0.00 g/L,0.26 g/L,0.40 g/L,1.40 g/L and 2.80 g/L.The human liver cells lines L-O2 in the exponential growth phase were cultured into 96-well plates,1 ×103 cells per well After 24 hours,the concentrations of amanita toxic peptides mentioned above were added.The minimum concentration of mushroom toxins keeping the liver cells alive was determined after 24,48 and 72 hours,respectively,and MTT method was used to test each group's liver cell activity.The experiment included 3 groups:the control group,the damage group,and the cholic acid group.Each group had 3 time points:24,48 and 72 hours after being attacked.Twenty four hours after attack,in cholic acid group,cholic acid drugs including taurocholic acid gca,goose deoxycholic acid,gansu ammonia goose deoxycholic acid and bovine goose deoxycholic acid were given,respectively,to protect the injured liver cells.Cells' morphology changes were observed under the inverted phase contrast microscope,living cells were counted by using MTT method,and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities in the culture supernatant were tested by the biochemical method.Results The minimum attack concentration of lamanita toxic peptides keeping liver cell survival in vitro was 1.40 g/L.Seventy-two hours after attack by amanita toxic peptides,the absorbance value was 0.812 ± 0.035,0.345 ± 0.021,0.363 ± 0.018,0.387 ± 0.027,0.431 ± 0.018,0.465 ± 0.015 and 0.452 ± 0.030,respectively in the control group,the damage group,the taurocholic acid group,the goose deoxycholic acid group,the glycocholic acid group,the glycochenodeoxycholic acid group and the sodium deoxycholic acid group.Compared with the damage group,absorbance value 72 hours after attack in each cholic acid group gradually increased,and compared with damage group,the differences were statistically significant among goose deoxycholic acid group,glycocholic acid group,glycochenodeoxycholic acid group and sodium deoxycholic acid group(P ＜ 0.05).AST and ALT activity in each cholic acid group declined,and that in glycochenodeoxycholic acid group was the lowest.Compared with the damage group,the difference was statistically significant (P ＜ 0.01).Conclusions Cholic acid can protect human liver cells from the damage induced by amanita toxic peptides.Such effect may be related to the fact that both amanita toxic peptides and cholic acid are the substrates of NTCP and OATP.