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Exosomes are cell-secreted membrane-coated vesicles with their sizes variable from 30 to 150 nm. So far, there is no simple, fast, and economical way to evaluate the sizes of exosomes in living systems. Here, we put forward a hypothesis in which the sphere sizes (resulting in different curvature) may affect the local mobility/viscosity of exosome membranes. Based on this hypothesis, we propose a novel method to evaluate the exosome sizes by quantifying the membrane viscosity. For this sake, we design a membrane-targeting molecular rotor with its fluorescence lifetime sensitive to viscosity and use it under a fluorescence lifetime imaging microscope (FLIM). Through a multiple-step ultrafiltration technique, we isolate three individual size distributions (10-50, 50-100, and 100-220 nm) with exosomes from HeLa and MCF-7 cell culture media and then perform the FLIM assay on the above two groups. In both cases, we indeed find a regular pattern in which the membrane viscosity reflected by lifetime decreases with exosome sizes. We then perform the assay on exosomes from cancer cells, corresponding normal tissue cells, and serum of breast cancer patients. We find that exosomes from cancer cells have a fluorescence lifetime (larger viscosity) longer than that of normal tissue cells. The average fluorescence lifetime of exosomes from a triple-negative breast cancer patient is longer (or the viscosity is larger) than that of a HER2 positive one. Therefore, our new and simple method may hold application prospects in future cancer diagnosis.

Objective Fatigue has become one of the major threats to human health in the 21st century. Traditional Chinese medicine (TCM), which proved to be safer and more effective, has become a hot spot in antifatigue research. Human placenta, also called “Ziheche”, has drawn great attention in the antifatigue effect since the Tang dynasty. However, the shortage of human placenta restricts wide usage of it. According to the theory of TCM, sheep placenta (SP) also has the effect of nourishing blood, tranquilization, nourishing skin, and prolongation of life. The aim of this study was to examine the antifatigue effects of sheep placenta peptide (SPP), an extract of sheep placenta, in mice and the mechanism was also studied. Methods Peptide from fresh SP was extracted via enzymolysis. SPP (0.13 g/kg) and soybean peptide (0.65 g/kg) were administrated orally and daily to mice for four weeks. Antifatigue effects of SPP were estimated on weight-loaded swimming test; A non-weight-loaded swimming test was conducted to elucidate underlying the mechanisms of the anti-fatigue effects. Results Administration of SPP prolonged the weight-loaded swimming time in mice. In addition, SPP significantly decreased the levels of muscle malondialdehyde (MDA) and serum lactic acid (LD), and increased the activities of muscle glutathione peroxidase (GSH), and superoxide dismutase (SOD) and liver glycogen in mice after non-weight-loaded swimming test. Moreover, the electron microscope observation showed that the muscle fiber bundle ranked neatly, the H band, I band, Z line as well as M line were clear and the numbers of mitochondria was normal though some of the mitochondria were swell in SPP treated mice after non-weight-loaded swimming test. Conclusion SPP possesses potent abilities for antifatigue; Increasing the anti-oxidant activities and energy reserve as well as improving the ultrastructures in gastrocnemius muscle cells, which may be the mechanisms of SPP exerting its antifatigue effects.

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.

Mitochondrial pH plays a pivotal role in the regulation of physiological process. Developing ratiometric fluorescent probes for real-time detection of mitochondrial pH fluctuation is still highly demanded yet challenging. Herein, we present a novel strategy to design a ratiometric probe (FDI) by broadening absorption spectrum (360–700 nm) of fluorescein fluorophore. Unsaturated dialkene/indole quaternary ammonium moiety has broadened absorption and emission spectrum of fluorescein fluorophore to provide ratiometric detection by CLSM in dual excitation/dual emission mode, as well as directionally accumulated in mitochondria in living cells. Superior to commercially mitochondrial tracker, Carboxy-SNARF, probe FDI exhibits excellent and fast cell-membrane permeability and mitochondrial-targetability. It has been demonstrated that FDI could permit real-time monitoring of pH alkalization of mitochondria stimulated by chloroquine. Owing to nondestructive process and reversible ratiometric response to pH, image acquisition can be repeated frequently to trace and monitor the time course of mitochondrial pH responses. It is clearly confirmed that FDI would be a promising probe for real-time tracking of mitochondrial pH changes in the biomedical and biological fields.

A ratiometric fluorescent sensor (Hoe-NI) was developed by connecting a nucleus targeted Hoechst unit to a naphthalimide dye via “click chemistry”. The sensor achieves high specific nucleus labeling with wash-free staining method in various kinds of living cells. The fluorescence ratio of the two emission bands (450 nm for Hoechst and 505 nm for naphthalimide) is changed sensitively to the variation of DNA concentrations, which provides the quantitative information in the processes of DNA damage induced by hydroxyl radicals and antitumor drug. Therefore, Hoe-NI is a recommendable sensor for the monitoring of nuclear DNA damage that reveals the health status of cells.

Viscosity, as one of the major factors of intracellular microenvironment, influences the function of proteins. To detect local micro-viscosity of a protein, it is a precondition to apply a viscosity sensor for specifically target to proteins. However, all the reported small-molecule probes are just suitable for sensing/imaging of macro-viscosity in biological fluids of entire cells or organelles. To this end, we developed a hybrid sensor BDP-V BG by connecting a viscosity-sensitive boron-dipyrromethene (BODIPY) molecular rotor (BDP-V) to O6-benzylguanine (BG) for specific detection of local micro-viscosity of SNAP-tag fused proteins. We measured and calculated the reaction efficiency between the sensor and SNAP-tag protein in vitro to confirm the high labeling specificity. We also found that the labeling reaction results in a 53-fold fluorescence enhancement for the rotor, which qualifies it as a wash-free sensor with ignorable background fluorescence. The high sensitivity of protein labeled sensor (BDP-V-SNAP) to the changes of local viscosity was evaluated by detecting the enhancement of fluorescence lifetimes. Further, with the sensor BDP-V BG, we achieved high specific labeling of cells expressing two SNAP-tag fused proteins (nuclear histone H2B and mitochondrial COX8A). Two-photon excited fluorescence lifetime imaging revealed that, the micro-viscosities nearby the SNAP-tag fused two proteins are distinct. The different changes of local micro-viscosity of SNAP-tag fused histone protein in apoptosis induced by three nucleus-targeted drugs were also characterized for the first time.

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Here we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003–0.005%) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3–100 μM), but not the other three, concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003–0.005%) and SJ-2 (0.3–100 μM) also inhibited ^Ca^ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003–0.001%) and SJ-2 (1–100 μM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells., 論文

It is meaningful to accurately quantify the changes in local viscosity within the mitochondria of living cells, because viscosity influences mitochondrial network organization and metabolite diffusion. Although many molecular probes targeting mitochondria have been reported, almost all of them are not fixed to the mitochondria. Thus, they may not be suitable for sensing in abnormal mitochondria with lowered potential. In order to monitor viscosity in all mitochondria, no matter their working or health status, we develop the first fixable BODIPY (boron-dipyrromethene) rotor, named Vis-A. Vis-A contains an aldehyde group as an anchor to react with amino groups of proteins, which make it immobilizable in mitochondria. Vis-B, the reference compound without such anchor unit, is also synthesized. Both Vis-A and Vis-Bshow excellent mitochondrial targetability, as good as the commercially available mitochondrial dye (Mito Tracker Deep Red). However, when cells are chemically treated to decrease the mitochondrial potential, only Vis-A continues emitting strong fluorescence in mitochondria, but the signals of Vis-B and Mito Tracker Deep Red completely disappear. This comparison confirms that Vis-A not only specifically localizes in mitochondria, but also can be stably retained there. Then, Vis-A is applied to detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A, the changes in viscosity under typical pathological conditions (i.e., treatment with rotenone and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) for mitochondria are monitored by FLIM.

Near-infrared (NIR) fluorescent probes are advantageous over visible ones, for they can avoid the interference from the short-wavelength background emission in biological systems. However, there are a very limited number of NIR probes that can specifically label target proteins in living cells. In this work, a series of long-wavelength dyes (N-NIR, S-NIR, and K-NIR) analogous to the novel Changsha NIR family are synthesized conveniently through a new approach that is different from the previously reported one. These three dyes have similar conjugation structures but exhibit tunable photophysical properties. N-NIR and S-NIR have large extinction coefficients over 100 000, and high fluorescence quantum yields. Although NIR absorption and emission of K-NIR are inferior to the former two, it emits in a much longer wavelength region. And all the three dyes can easily pass through the cell membranes to obtain the high-resolution NIR fluorescence images. Furthermore, N-NIR is chosen as the NIR fluorophore to develop a protein-labeling reagent PYBG-D, since it demonstrates the highest fluorescence quantum yield of up to 0.4 (in methanol). PYBG-D is efficiently synthesized through Sonogashira coupling between bromo-substituted N-NIR and alkyne-substituted benzylguanine (PYBG). The conjugate PYBG-D proves to be a specific and efficient label for O6-alkylguanine-DNA alkyltransferase (SNAP-tag) that fused to target proteins in living cells, which contributes to high resolution NIR fluorescence images under a laser confocal microscope.

By connection of O(6)-benzylguanine (BG) to an "o-phenylenediamine-locked" rhodamine spirolactam responsive to nitric oxide (NO), a novel substrate (TMR-NO-BG) of genetically encoded SNAP-tag has been constructed. In living cells, labeling SNAP-tag fused proteins with TMR-NO-BG will in situ generate corresponding probe-protein conjugates (TMR-NO-SNAP) that not only inherit high NO sensitivity from the small-molecule parent but also guarantee the site-specificity to the designated subcellular compartments such as the mitochondrial inner membrane, nucleus, and cytoplasm. In two representative cellular processes, TMR-NO-BG demonstrates its applicability to monitor endogenous subcellular NO in the activated RAW264.7 cells stimulated by lipopolysaccharide and in the apoptotic COS-7 cells induced by etoposide.

We have developed two near-infrared fluorescent probes for pH, named pH-A and pH-B. The fluorescence quantum yields of pH-A and pH-B are 0.19 and 0.095 in methanol. And there is the neutral pKa value of pH-A (pKa = 7.2). What is the most important is that these probes can stain the cells, which can reflect the changes of pH in cells.

Glycyrrhiza Uralensis Polysaccharide (GCP), as a macromolecular polysaccharide extracted from the Traditional Chinese Medicine (TCM) - Licorice has been proved to inhibit tumor growth in vitro and in vivo; however, the specific anti-tumor mechanism of GCP needs to be further investigated. In this study, we explore the anti-tumor mechanism of GCP from the angle of gut microbiota. Colon carcinoma cells (CT-26) were used to set up a tumor-bearing mouse model. After 14 days of GCP treatment, the weights of tumors were significantly reduced. In addition, HE staining of tissue sections reflected that GCP could effectively inhibit tumor metastasis. 16SrRNA high-throughput sequencing of fecal samples showed a significant change between the model group and GCP group in the composition of gut microbiota. Subsequently, gut microbiota depletion and fecal transplantation experiments further confirmed the relationship between the anti-tumor effects of GCP and gut microbiota. Following depletion of gut microbiota, GCP cannot inhibit tumor growth. Fecal transplantation experiments found that transplanting the feces of GCP-treated mice, to a certain extent, could inhibit tumor growth and metastasis. These results indicate that Glycyrrhiza Polysaccharides exert anti-tumor effects by affecting gut microbiota composition. Keywords: Glycyrrhiza Uralensis Polysaccharides, Gut microbiota, Cancer, TCM, Anti-tumor

Genetically encoded technologies provide methods for specific labeling and imaging of proteins, which is essential for the understanding of their subcellular localization and function. Herein, we employ naphthalimide, an efficient two-photon fluorophore, to develop O6-benzylguanine (BG) derivatives for specific labeling of subcellular proteins and fluorescent imaging through the SNAP-tag. The three naphthalimide-BG derivatives, TNI-BG, QNI-BG and ONI-BG are conveniently synthesized through modular "click chemistry" with high yields. All of them show high labeling efficiency with SNAP-tag in solution (1~2 ×103 s-1 M-1) and in bacteria. Among them, ONI-BG shows high specificity to diffused, histone H2B and mitochondria COX8A targeted SNAP-tag in mammalian cells. The protein labeled naphthalimides exhibit high two-photon absorption cross section, which indicates the potential application in protein specific two-photon fluorescent imaging, such as two-photon fluorescent lifetime imaging and two-photon multi-color imaging. Therefore, ONI-BG is a versatile tool for tracking of subcellular proteins via multiple fluorescent techniques.