Hypochlorite (ClO-) and viscosity both affect the physiological state of mitochondria, and their abnormal levels are closely related to many common diseases. Therefore, it is vitally important to develop mitochondria-targeting fluorescent probes for the dual sensing of ClO- and viscosity. Herein, we have explored a new fluorescent probe, XTAP-Bn, which responds sensitively to ClO- and viscosity with off-on fluorescence changes at 558 and 765 nm, respectively. Because the emission wavelength gap is more than 200 nm, XTAP-Bn can effectively eliminate the signal crosstalk during the simultaneous detection of ClO- and viscosity. In addition, XTAP-Bn has several advantages, including high selectivity, rapid response, good water solubility, low cytotoxicity, and excellent mitochondrial-targeting ability. More importantly, probe XTAP-Bn is successfully employed to monitor the dynamic change in ClO- and viscosity levels in the mitochondria of living cells and zebrafish. This study not only provides a reliable tool for identifying mitochondrial dysfunction but also offers a potential approach for the early diagnosis of mitochondrial-related diseases.

Aryl Hydrocarbon Receptor (AHR) ligands, upon binding, induce distinct gene expression profiles orchestrated by the AHR, leading to a spectrum of pro- or anti-inflammatory effects. In this study, we designed, synthesized and evaluated three indole-containing potential AHR ligands (FluoAHRL: AGT-4, AGT-5 and AGT-6). All synthesized compounds were shown to emit fluorescence in the near-infrared. Their AHR agonist activity was first predicted using in silico docking studies, and then confirmed using AHR luciferase reporter cell lines. FluoAHRLs were tested in vitro using mouse peritoneal macrophages and T lymphocytes to assess their immunomodulatory properties. We then focused on AGT-5, as it illustrated the predominant anti-inflammatory effects. Notably, AGT-5 demonstrated the ability to foster anti-inflammatory regulatory T cells (Treg) while suppressing pro-inflammatory T helper (Th)17 cells in vitro. AGT-5 actively induced Treg differentiation from naïve CD4+ cells, and promoted Treg proliferation, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) expression and interleukin-10 (IL-10) production. The increase in IL-10 correlated with an upregulation of Signal Transducer and Activator of Transcription 3 (STAT3) expression. Importantly, the Treg-inducing effect of AGT-5 was also observed in human tonsil cells in vitro. AGT-5 showed no toxicity when applied to zebrafish embryos and was therefore considered safe for animal studies. Following oral administration to C57BL/6 mice, AGT-5 significantly upregulated Treg while downregulating pro-inflammatory Th1 cells in the mesenteric lymph nodes. Due to its fluorescent properties, AGT-5 could be visualized both in vitro (during uptake by macrophages) and ex vivo (within the lamina propria of the small intestine). These findings make AGT-5 a promising candidate for further exploration in the treatment of inflammatory and autoimmune diseases.

Background: Osteoarthritis (OA) standing as the most prevalent form of arthritis, closely associates with heightened levels of reactive oxygen species, particularly hypochlorous acid (HOCl). Although there are numerous probes available for detecting HOCl in the OA region, probes with dual functions of diagnostic and therapeutic capabilities are still significantly lacking. While this type of probe can reduce the time gap between diagnosis and treatment, which is clinically needed. Methods: We developed a fluorescent probe (DHU-CBA1) toward HOCl with theranostics functions through the release of methylene blue (MB) and ibuprofen (IBP) in this work. DHU-CBA1 can detect HOCl with high specificity and sensitivity, releasing MB and IBP with an impressive efficiency of ≥ 95% in vitro. Results: DHU-CBA1 exhibits good biosafety, enabling in vivo imaging of endogenous HOCl, along with reducing arthritis scores, improving synovitis and cartilage damage, and maintaining catabolic balance while alleviating senescence in cartilage. Conclusions: This study proposes a novel approach to enhance osteoarthritis therapy by releasing IBP via a smart HOCl-enabled fluorescent probe.

A CHA-based fluorescent DNA tetrahedral probe (FDTp) has been designed to detect the microRNAs miR-21 and miR-155 sensitively and specifically in living cells. The design consisted of functional elements (H1, H2, and Protector) connected to a DNA tetrahedron modified with two pairs of fluorophores and quenching groups. In the presence of miR-21, the chain displacement effect was triggered and Cy3 fluorescence was emitted. In the presence of miR-155, the signal of the catalytic hairpin assembly (CHA) between H1 and H2 on FDTp was amplified, making the fluorescence of FAM sensitive to miR-155. Using this method, the detection limit for miR-155 was 5 pM. The FDTp successfully imaged miR-21 and miR-155 in living cells and distinguished a variety of cell lines based on their expression levels of miR-21 and miR-155. The detection and imaging of dual targets in this design ensured the accuracy of tumor diagnosis and provided a new method for early tumor diagnosis.

OBJECTIVE: Studies on the appropriate amount of anti-adhesive agents for preventing postoperative adhesion are lacking. This animal study aimed to investigate the distribution of an anti-adhesive agent in the abdominal cavity and estimate the necessary amount to cover the entire cavity.
METHODS: Fluorescent dye Flamma-552 was conjugated to Guardix-sol to create Guardix-Flamma, which was laparoscopically applied to the abdominal cavity of two 10-kg pigs in different amounts: 15 mL for G1 and 35 mL for G2. After 24 hours, the distribution of Guardix-Flamma was examined under the near-infrared mode of the laparoscope, and the thickness was measured in tissues from the omentum, small, and large intestine by immunohistochemistry.
RESULTS: The average area of the abdominal cavity in 10 kg pigs was 2,755 cm2. Guardix-Flamma fluorescence was detected in the greater omentum, ascites in the pelvis, and right quadrant area in G1, whereas in G2, it was detected everywhere. On average, the total thickness of G1 and G2 were 12.68 ± 9.80 μm and 18.16 ± 15.57 μm, respectively. Guardix-Flamma thickness applied to the omentum, small, and large intestines of G2 were 1.31-, 1.45-, and 1.49-times thicker than those of G1, respectively, and were all statistically significant (P < 0.05).
CONCLUSIONS: The entire abdominal cavity of the 10 kg pig was not evenly covered with 15 mL of Guardix. Although 35 mL of Guardix is sufficient to cover the same area with an average thickness of 18 µm, further studies should evaluate the minimum thickness required for an effective anti-adhesive function.

2-Hydroxyoleic acid (2-OHOA) has gained attention as a membrane lipid therapy (MLT) anti-cancer drug. However, in the viewpoint of anti-cancer drug, 2-OHOA shows poor water solubility and its effectiveness still has space for improvement. Thus, this study aimed to overcome the problems by formulating 2-OHOA into liposome dosage form. Furthermore, in the context of MLT reagents, the influence of 2-OHOA on the biophysical properties of the cytoplasmic membrane remains largely unexplored. To bridge this gap, our study specifically focused the alterations in cancer cell membrane fluidity and lipid packing characteristics before and after treatment. By using a two-photon microscope and the Laurdan fluorescence probe, we noted that liposomes incorporating 2-OHOA induced a more significant reduction in cancer cell membrane fluidity, accompanied by a heightened rate of cellular apoptosis when compared to the non-formulated 2-OHOA. Importantly, the enhanced efficacy of 2-OHOA within the liposomal formulation demonstrated a correlation with its endocytic uptake mechanism. In conclusion, our findings underscore the significant influence of 2-OHOA on the biophysical properties of cancer plasma membranes, emphasizing the potential of liposomes as an optimized delivery system for 2-OHOA in anti-cancer therapy.

The analysis of recombinant proteins in complex solutions is often accomplished with tag-specific antibodies in western blots. Recently, I introduced an antibody-free alternative wherein tagged proteins are visualized directly within polyacrylamide gels. For this, I used the protein ligase Connectase to selectively attach fluorophores to target proteins possessing an N-terminal recognition sequence. In this study, I extend this methodology to encompass the detection and quantification of C-terminally tagged proteins. Similar to the N-terminal labeling method, this adapted procedure offers increased speed, heightened sensitivity, and an improved signal-to-noise ratio when compared to western blots. It also eliminates the need for sample-specific optimization, enables more consistent and precise quantifications, and uses freely available reagents. This study broadens the applicability of in-gel fluorescence detection methods and thereby facilitates research on recombinant proteins.

Intracellular Ca2+ can be conveniently monitored by sensitive Ca2+ fluorescent dyes in live cells. The Gαq involved lipid signaling pathways and, thus, can be studied by intracellular Ca2+ imaging. Here we describe the protocols to measure intracellular Ca2+ for studying PEG2-EP1 activity in esophageal smooth muscle cells. The ratiometric Fura-2 imaging provides quantitative data, and the Fluo-4 confocal microscopic imaging has high-spatial resolution.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

Seed germination of a parasitic plant Striga hermonthica is elicited by strigolactones which are exuded from roots of host plants. Here, we describe a high-throughput germination assay and a method for visualizing in vivo strigolactone receptor functions with a fluorogenic probe.