Super-Resolution Imaging of Autophagy by a Preferred Pair of Self-Labeling Protein Tags and Fluorescent Ligands.

Autophagy is a core recycling process for homeostasis, with its dysfunction associated with tumorigenesis and various diseases. Yet, its subtle intracellular details are covered due to the limited resolution of conventional microscopies. The major challenge for modern super-resolution microscopy deployment is the lack of a practical labeling system, which could provide robust fluorescence with fidelity in the context of the dynamic autophagy microenvironment. Herein, a representative autophagy marker LC3 protein is selected to develop two hybrid self-labeling systems with tetramethylrhodamine (TMR) fluorophores through SNAP/Halo-tag technologies. A systematic investigation indicated that the match of the LC3-Halo and TMR ligand remarkably outperforms that of LC3-SNAP, as the former Halo system exhibited more robust single-molecule brightness (440 vs 247), total photon numbers (45600 vs 13500), and dwell time of the initial bright state (0.82 vs 0.40 s) than the latter. With the aid of this desirable Halo system, for the first time, live-cell ferritinophagy is monitored with a spatial resolution of ∼50 nm, which disclosed reduced sizes of autophagosomes (∼650 nm, ferritinophagy) than those in nonselective (∼840 nm, mammalian target of rapamycin (mTOR)) and selective autophagy (∼900 nm, mitophagy).

Metabolomics-guided analysis reveals a two-step epimerization of deoxynivalenol catalyzed by the bacterial consortium IFSN-C1.

Deoxynivalenol (DON) is commonly found in wheat and wheat-derived foods, posing a threat to human health. Biodegradation is an efficient and eco-friendly measure for mycotoxin detoxification. Understanding the mechanism of DON biodegradation is hence of great importance. Herein, we report the application of metabolomics methods for the analysis of DON degradation by a bacterial consortium isolated from wheat leaves collected in Jiangsu Province. Metabolomics analysis combined with a nuclear magnetic resonance analysis revealed the main degradation product, 3-keto-DON, and a minor degradation product, 3-epi-DON. Further study illustrated that DON underwent a two-step epimerization through the intermediate 3-keto-DON. Sequencing analysis of the 16S rRNA metagenome of the microorganismal community suggested that the abundance of three bacterial genera, Achromobacter, Sphingopyxis, and Sphingomonas, substantially increased during the coculture of bacterial consortium and DON. Further investigation revealed that Devosia sp. might be responsible for the epimerization of 3-keto-DON. These findings shed light on the catabolic pathways of DON during biodegradation and illustrate the potential of using metabolomics approaches in biodegradation studies.Key Points• A bacterial consortium was isolated with good deoxynivalenol-degrading potential. • Metabolomics approaches were successfully used to interpret the degradation pathway. • A trace-amount degradation product was determined by metabolomics and NMR analysis. Graphical Abstract .

Cytotoxic Trichothecene Macrolides Produced by the Endophytic Myrothecium roridum.

Six new macrolides named myrothecines D-G (1-4), 16-hydroxymytoxin B (5), and 14'-dehydrovertisporin (6), including four 10,13-cyclotrichothecane derivatives, in addition to 12 known compounds (7-18), were isolated from three endophytic Myrothecium roridum, IFB-E008, IFB-E009, and IFB-E012. The isolated compounds were characterized by MS, NMR, CD, and single-crystal X-ray crystallography. The isolated macrolides exhibited an antiproliferation effect against chronic myeloid leukemia K562 and colorectal carcinoma SW1116 cell lines. Compounds 1-6 were cytotoxic, with IC50 values ranging between 56 nM and 16 µM. Since slight structural changes led to obvious activity differences, the CoMFA (comparative molecular field analysis) and CoMSIA (comparative molecular similarity indices analysis) methods were then used to explore the 3D QSAR (three-dimensional quantitative structure-activity relationship) of these macrolides. The result showed that the steric, electrostatic, hydrophobic, and H-bond acceptor factors were involved in their cytotoxicity and provided an in-depth understanding of the structure-activity relationships of these metabolites.

A Mechanism-Based Model for the Prediction of the Metabolic Sites of Steroids Mediated by Cytochrome P450 3A4.

Early prediction of xenobiotic metabolism is essential for drug discovery and development. As the most important human drug-metabolizing enzyme, cytochrome P450 3A4 has a large active cavity and metabolizes a broad spectrum of substrates. The poor substrate specificity of CYP3A4 makes it a huge challenge to predict the metabolic site(s) on its substrates. This study aimed to develop a mechanism-based prediction model based on two key parameters, including the binding conformation and the reaction activity of ligands, which could reveal the process of real metabolic reaction(s) and the site(s) of modification. The newly established model was applied to predict the metabolic site(s) of steroids; a class of CYP3A4-preferred substrates. 38 steroids and 12 non-steroids were randomly divided into training and test sets. Two major metabolic reactions, including aliphatic hydroxylation and N-dealkylation, were involved in this study. At least one of the top three predicted metabolic sites was validated by the experimental data. The overall accuracy for the training and test were 82.14% and 86.36%, respectively. In summary, a mechanism-based prediction model was established for the first time, which could be used to predict the metabolic site(s) of CYP3A4 on steroids with high predictive accuracy.

A Golgi-targeted viscosity rotor for monitoring early alcohol-induced liver injury.

We proposed to monitor the early stage of alcohol-induced liver injury through quantitatively detecting Golgi viscosity. Therefore, the first Golgi-targeted fluorescent rotor (GA-Vis) was developed. With the aid of GA-Vis, the changes in Golgi viscosity during alcohol-induced liver injury were quantitatively evaluated by fluorescence lifetime imaging in live cells and zebrafish. GA-Vis was qualified as a practical tool for future diagnoses of alcohol-induced liver injury.

Hybrid labeling system for dSTORM imaging of endoplasmic reticulum for uncovering ultrastructural transformations under stress conditions.

The endoplasmic reticulum (ER) transforms its morphology to fit versatile cellular functions especially under stress conditions. Since various ER stresses are critical pathophysiological factors, the precise observations of ER can provide insights into disease diagnoses and biological researches. Live-cell super-resolution imaging is highly expected for uncovering microstructures of ER. However, to achieve this, there remains a big challenge in how to efficiently label ER with advanced fluorophores. Herein, we report a new SNAP-tag fluorescent probe, namely, CLP-TMR, for specific and high-density labeling of the newly constructed dual ER-signal (targeting and retention) peptides fused-SNAP proteins. This hybrid labeling system integrating chemical probes with genetically encoded techniques enables molecular position reconstructions of ER morphologies through direct stochastic optical reconstruction microscopy (dSTORM) imaging. The super-resolution imaging reveals several never-known ultrastructural changes responding to different ER stresses, i.e. the formation of peripheral ER sheets to restore the immunogenicity, and the long flattened ER tubules under inflammation.

A targetable fluorescent probe for dSTORM super-resolution imaging of live cell nucleus DNA.

HoeSR, a nucleus specific probe for dSTORM super-resolution imaging of nucleus DNA in live cells, was designed by conjugating a rhodamine fluorophore and a Hoechst tag. HoeSR labels the cell nucleus in a wash-free way and emits intensive fluorescence exclusively in the nucleus. With the aid of HoeSR, nucleus nanostructures at different mitosis stages were observed through super-resolution imaging.

Quantitatively monitoring oxygen variation in endoplasmic reticulum with a fluorophore-phosphor energy transfer cassette.

An efficient excitation energy transfer (EET) cassette, i.e.Ir-Np-OH, has been constructed by connecting an Ir(iii) complex phosphor as an acceptor to a naphthalimide fluorophore as a donor. Ir-Np-OH emits dual emissions, with one highly dependent on oxygen concentration and the other completely independent. Thus, Ir-Np-OH exhibits a sensitive and quantitative response to oxygen level in a ratiometric way. The linear response of Ir-Np-OH ranges from 0% (0 mmHg) to 10% (76 mmHg) oxygen tension, which matches very well with intracellular oxygen levels. And the ratiometric oxygen sensing of Ir-Np-OH is completely reversible. Also, importantly, Ir-Np-OH possesses a subcellular targetability specifically toward endoplasmic reticulum (ER), as proved by the co-localization imaging experiment. This feature of Ir-Np-OH should be noted, because generally speaking, it is much more challenging for small-molecule probes to target ER than other subcellular organelles, e.g. mitochondria. By using Ir-Np-OH, an apparent increase in oxygen level from 5.17% (39.29 mmHg) to 7.29% (55.40 mmHg) is monitored in ER of live MCF-7 cells, during the 30 minute stimulation with Rotenone, a typical inhibitor of the mitochondrial respiratory chain. In addition, upon ER stress directly induced by TG (Thapsigargin), the gradual increase in oxygen concentration is also clearly detected. These results confirm that Ir-Np-OH is a promising tool for oxygen-related cellular and chemical biology.

Specific Recognition of Breast Cancer Cells In Vitro Using Near Infrared-Emitting Long-Persistence Luminescent Zn3Ga2Ge2O10:Cr3+ Nanoprobes.

In this paper, near-infrared emitting long-persistence luminescent Zn3Ga2Ge2O10:Cr3+ (ZGG) nanoparticles with diameters of 30-100 nm and bright luminescence were prepared by a sol-gel synthesis method. After the surface amination, the nanoparticles were further bioconjugated with breast cancer-specific monoclonal antibody (anti-EpCAM) to form ZGG-EpCAM nanoprobes which can specifically target breast cancer cell lines (MCF7) in vitro. The results of in vitro images show that the luminescence signals from the cells treated with ZGG-EpCAM nanoprobes are stronger than those from cells treated with ZGG-unconjugated antibody, indicating that the prepared ZGG-EpCAM nanoprobes possessed excellent specific recognition capability. Furthermore, due to their long afterglow properties, the imaging could persist more than 1 h. Therefore, these nanoprobes could not only provide a high specificity detection method for cancer cells but also realize the long-time monitoring. Developed near-infrared emitting long-persistence luminescent nanoprobes will be expected to find new perspectives for cell therapy research and diagnosis applications.

Multifunctional near infrared-emitting long-persistence luminescent nanoprobes for drug delivery and targeted tumor imaging.

In this paper, near infrared-emitting long-persistence luminescent porous Zn1.1Ga1.8Ge0.1O4:Cr(3+), Eu(3+) @SiO2 nanoprobes have been prepared using mesoporous silica nanospheres both as morphology-controlling templates and as vessels. These nanoprobes possessed an excellent capacity for drug delivery and allowed for real-time monitoring of the delivery routes of the drug carriers in vivo. The nanoprobes demonstrated a typical mesoporous structure, a brighter NIR emission (696 nm) and a long afterglow luminescence that persisted for 15 d. Furthermore, after surface modification with folic acid (FA), a tumor-targeting group, these nanoprobes exhibited an excellent ability to target tumors with high sensitivity in vitro and in vivo. Importantly, these modified nanoprobes could accurately diagnose tumors and allow for long-term tumor monitoring via in situ and in vivo re-excitation by a red LED lamp. Furthermore, the drug release data demonstrated that the modified nanoprobes could be loaded with a large amount of doxorubicin hydrochloride (DOX) and showed sustained release behavior. Together, the results of this study indicate that these nanoprobes can accurately diagnose tumors, allow for long-term in vivo and in situ monitoring and release DOX in situ to cure tumors.