Probe oscillation control in tapping-mode scanning probe electrospray ionization for stabilization of mass spectrometry imaging.

Mass spectrometry imaging (MSI) is used for visualizing the distribution of components in solid samples, such as biological tissues, and requires a technique to ionize the components from local areas of the sample. Tapping-mode scanning probe electrospray ionization (t-SPESI) uses an oscillating capillary probe to extract components from a local area of a sample with a small volume of solvent and to perform electrospray ionization of those components at high speed. MSI can be conducted by scanning the sample surface with a capillary probe. To ensure stable extraction and ionization for MSI, the probe oscillation during measurements must be understood. In this study, we examined the changes in oscillation amplitude and phase due to the interaction between the oscillating probe and the brain tissue section when the probe tip was dynamically brought close to the sample surface. The changes in the probe oscillation depended on the oscillation frequency and polarity of the bias voltage applied to the solvent because an electrostatic force shifted the frequency of the probe oscillation. These findings suggest that controlling the probe oscillation frequency is important for stabilizing MSI by t-SPESI.

Investigation of Peptide Labeling with ortho-Phthalaldehyde and 2-Acylbenzaldehyde.

ortho-Phthalaldehyde (OPA) with high reactivity to the amine group has been widely used to modify proteins. We discovered new modifications of OPA and 2-acylbenzaldehyde and proposed the reaction mechanism. Using isotope labeling mass spectrometry-based experiment, we identified new cross-linking properties of OPA and 2-acylbenzaldehyde. This reactivity revealed that OPA has the potential to probe proximal amino acids in biological systems.

Solvent effects of N,N-dimethylformamide and methanol on mass spectrometry imaging by tapping-mode scanning probe electrospray ionization.

Mass spectrometry imaging (MSI) is an effective technique for visualizing the distribution of lipids in tissues. The direct extraction-ionization methods using minute volumes of solvent for local components have the advantage of rapid measurement without any sample pretreatment. For effective MSI of tissues, it is necessary to understand the effect of solvent physicochemical properties on ion images. In this study, we report solvent effects on the lipid imaging of mouse brain tissue by tapping-mode scanning probe electrospray ionization (t-SPESI) which is capable of extraction-ionization using sub-pL solvents. To precisely measure lipid ions, we developed a measurement system incorporating a quadrupole-time-of-flight mass spectrometer. The differences in signal intensity and spatial resolution of lipid ion images were investigated using N,N-dimethylformamide (non-protic polar solvent), methanol (protic polar solvent) and their mixture. The mixed solvent was suitable for the protonation of lipids, and it provided high spatial resolution MSI. Results indicate that the mixed solvent improves the extractant transfer efficiency and minimizes charged droplets from an electrospray. The solvent selectivity study revealed the importance of solvent selection based on physicochemical properties for the advancement of MSI by t-SPESI.

Biological Evaluation of 8-Methoxy-2,5-dimethyl-5H-indolo[2,3-b] Quinoline as a Potential Antitumor Agent via PI3K/AKT/mTOR Signaling.

Chemotherapy is commonly used clinically to treat colorectal cancer, but it is usually prone to drug resistance, so novel drugs need to be developed continuously to treat colorectal cancer. Neocryptolepine derivatives have attracted a lot of attention because of their good cytotoxic activity; however, cytotoxicity studies on colorectal cancer cells are scarce. In this study, the cytotoxicity of 8-methoxy-2,5-dimethyl-5H-indolo[2,3-b] quinoline (MMNC) in colorectal cells was evaluated. The results showed that MMNC inhibits the proliferation of HCT116 and Caco-2 cells, blocks the cell cycle in the G2/M phase, decreases the cell mitochondrial membrane potential and induces apoptosis. In addition, the results of western blot experiments suggest that MMNC exerts cytotoxicity by inhibiting the expression of PI3K/AKT/mTOR signaling pathway-related proteins. Based on these results, MMNC is a promising lead compound for anticancer activity in the treatment of human colorectal cancer.

Subcellular Interactomes Revealed by Merging APEX with Cross-Linking Mass Spectrometry.

Subcellular protein-protein interactions (PPIs) are essential to understanding the mechanism of diverse cellular signaling events and the pathogenesis of diseases. Herein, we report an integrated APEX proximity labeling and chemical cross-linking coupled with mass spectrometry (CXMS) platform named APEX-CXMS for spatially resolved subcellular interactome profiling in a high-throughput manner. APEX proximity labeling rapidly captures subcellular proteomes, and the highly reactive chemical cross-linkers can capture weak and dynamic interactions globally without extra genetic manipulation. APEX-CXMS was first applied to mitochondria and identified 653 pairs of interprotein cross-links. Six pairs of new interactions were selected and verified by coimmunoprecipitation, the mammalian two-hybrid system, and surface plasmon resonance method. Besides, our approach was further applied to the nucleus, capturing 336 pairs of interprotein cross-links with approximately 94% nuclear specificity. APEX-CXMS thus provides a simple, fast, and general alternative to map diverse subcellular PPIs.

Synthesis of 8-Fluoroneocryptolepine and Evaluation for Cytotoxic Activity against AGS Cancer Cells.

Neocryptolepine derivatives have attracted great interest because of their unique cytotoxic activity. 8-Fluoroneocryptolepine (8FNC) was synthesized, and its cytotoxicity was evaluated by MTT assay in AGS gastric cancer cells and gastric mucosa GES-1 cells. 8-Fluoroneocryptolepine showed greater selectivity and cytotoxicity to AGS cells than the cisplatin (CIS) and fluorouracil (5-Fu) commonly used in clinical treatment of gastric cancer. Most importantly, we significantly improved the cytotoxic effect of 8FNC against AGS cells by structural modification and reduced the cytotoxicity against GES-1 cells compared with neocryptolepine. We further evaluated the activity of 8FNC against AGS cells in vitro. Our results indicate that 8FNC arrests the AGS cell cycle in the G2/M phase, reduces the mitochondrial membrane potential of AGS cells, and drives the initiation of apoptotic body formation in 8FNC-induced apoptosis. Moreover, 8FNC exhibits strong inhibitory effects on AGS cell migration. Studies on the molecular mechanisms of the cytotoxic activities of 8FNC revealed that it may play a significant role in the inhibitory effect on AGS human gastric cancer cells through the PI3K/AKT signaling pathway. In conclusion, 8FNC may become a promising lead compound in the development of potential clinical drug candidates for the treatment of gastric cancer.

Natural Environmental Variation Determines Microbial Diversity Patterns in Serofluid Dish, a Traditional Chinese Fermented Vegetable Food.

Serofluid dish is a traditional fermented food that contains rich microbial populations. To gain insight into the environmental variables shaping the microbial diversity patterns, serofluid dish samples were collected from different areas, and 16S rRNA sequencing was performed. Analyses revealed both species and community diversity, including phylotype richness, Shannon index and phylogenetic diversity, were mostly influenced by pH. Additionally, such effects were corroborated by the Mantel test of pairwise UniFrac distances and variable selection of multiple linear regression models. Eventually, correlations between dominant lineages and the pH of serofluid dish other than geographical distance explained a large portion of the changes in microbial composition and diversity. Lactobacillus and related genera, Pediococcus and Acetobacter were largely driven by the variability of pH, and higher richness was observed under moderate pH ranges. Collectively, the results demonstrated that a microbial diversity pattern in serofluid dish is predictable by natural environmental variation and can be better understood through pH conditions.

Protocol for scarless genome editing of human pluripotent stem cell based on orthogonal selective reporters.

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and disease modeling. Here, we present a protocol for establishing edited hPSC cell lines utilizing visualized orthogonal selective reporters. We describe steps for constructing plasmids, carrying out cell culture and electroporation, as well as performing drug-fluorescent dual enrichment, clone screening, and cell line characterization. This protocol facilitates the achievement of single-base homozygous mutations and reporter knockins, offering a reliable approach for precision genome editing.

Anti-Gastric Cancer Activity of the Cell-free Culture Supernatant of Serofluid Dish and Lactiplantibacillus plantarum YT013.

Cancer is second only to heart disease as a cause of death, despite improvements in its early diagnosis and precision medicine. Due to the limitations of commonly used anticancer methods such as surgery, radiotherapy and chemotherapy, biological therapy, especially probiotics such as lactic acid bacteria, has received widespread attention. Lactobacillus has been proven to inhibit the proliferation of a variety of cancer cells. In this work, the effects of the cell-free culture supernatant of serofluid dish (CCS1) and the cell-free culture supernatant of Lactiplantibacillus plantarum YT013 (CCS2) isolated from serofluid dish on AGS, HCT116, HepG2 and PANC-1 cells were investigated. Based on the CCK-8 assay, CCS1 and CCS2 were shown to suppress the growth of cancer cells in a concentration-dependent manner. The IC50 values of CCS2 of AGS, HCT116, HepG2 and PANC-1 cells were 346.51 ± 35.28, 1207.69 ± 333.18, 650.94 ± 123.78 and 808.96 ± 126.27 µg/ml, respectively. In addition, the results of fluorescence microscopy showed that CCS2 changed cell morphology and treated with CCS2 (200, 400 and 800 µg/ml) for 48 h, AGS cell apoptosis was quantitatively surveyed by flow cytometry, showing 25.0, 34.1, and 42.6% total apoptotic cells. Moreover, western blotting confirmed that BAX, BAD and Caspase-3/8/9 were significantly upregulated and that BCL-2 was significantly downregulated in AGS cells treated with CCS2. These results indicated that CCS2 might lead to apoptosis via the endogenous mitochondrial apoptotic pathway. In summary, Lactiplantibacillus plantarum YT013 may be considered a good candidate for anticancer therapies.

Synthesis and anticancer evaluations of novel 1H-imidazole [4,5-f][1,10] phenanthroline derivative for the treatment of colorectal cancer.

1H-imidazole [4,5-f][1,10] phenanthroline is a promising chemical structure for cancer treatment. Herein, we synthesized a novel 1H-imidazole [4,5-f][1,10] phenanthroline derivative named IPM714 and found it exhibited selectively colorectal cancer (CRC) cells inhibitory activities, with half maximal inhibitory concentration (IC50) of 1.74 µM and 2 µM in HCT116 cells and SW480 cells, respectively. The present study is intended to explore the cytotoxicity of IPM714 in cancer cells of various types and its anticancer mechanism in vitro. Cellular functional analyses indicated IPM714 can arrest HCT116 cell cycle in S phase and induce apoptosis in HCT116 and SW480 cells. Western blot and molecular docking showed that IPM714 may suppress PI3K/AKT/mTOR pathway to inhibit cell proliferation and regulate cell cycle as well as apoptosis. This study proved IPM714 to be a promising drug in CRC therapy.

Synthesis and biological activity of 1H-imidazo[4,5-f][1,10]phenanthroline as a potential antitumor agent with PI3K/AKT/mTOR signaling.

1H-imidazo[4,5-f][1,10]phenanthroline (IPM713) is a type of tricyclic conjugated rigid planar structure with potential medical applications, but its anticancer activity has not yet been fully studied. In the present research, cells from seven different cancer types were used to study the anticancer effect, and IPM713 was found to inhibit the colorectal cancer cell line HCT116 most significantly, with a half maximal inhibitory concentration (IC50) of 1.7 µM. The mechanisms by which IPM713 exerts anti-colorectal cancer activity were studied. IPM713 blocked the cell cycle in G0/G1 phase and induced apoptosis by suppressing the PI3K/AKT/mTOR axis. In addition, an acute toxicity test showed that the median lethal dose (LD50) was above 5000 mg/kg. The findings of this research suggest that IPM713 can interfere with the PI3K/AKT/mTOR signaling pathway and might be a potential therapeutic candidate for the treatment of colorectal cancer.

Ultrasensitive assay based on a combined cascade amplification by nicking-mediated rolling circle amplification and symmetric strand-displacement amplification.

MicroRNAs (miRNAs) play an important role in the regulation of various biological processes and have been used as potential biomarkers for biomedical research and clinical diagnosis. Here, nicking-mediated rolling circle amplification (N-RCA)/symmetric isothermal circular strand-displacement amplification (S-SDA)-integrated combined cascade amplification (rs-CCA) was proposed for let-7a miRNA detection. Via introducing a palindromic fragment-integrated recognition sites for nicking endonuclease Nt.AlWI into the padlock probe, the hybridization of target miRNA can induce N-RCA and continuously generate the nicked fragments (NFs). Because the released NFs each have a palindromic sequence and daughter nicking site at the 3' end, they hybridize with each other, followed by alternately extension by polymerase and nicking by endonuclease. This leads to S-SDA effect, and the released single-stranded products in turn hybridize with NFs, accomplishing the rs-CCA process. When the Sybr Green I dyes intercalate into double-stranded DNA products, the amplified fluorescence signal is achieved. Thus, the target miRNA can be detected down to 5 pM. Importantly, the rs-CCA system is capable of distinguishing the single base difference between target miRNAs, indicating the high sequence specificity. Moreover, its potential application in disease diagnosis was demonstrated via detecting target miRNA in complex biological matrix and analyzing the total RNAs extracted from HeLa cells. As a proof-of-concept building, the impressive rs-CCA scheme is expected to provide a valuable insight into constructing powerful signal amplification strategies via sophisticated combination of biotechnologies available for nucleic acid manipulation and significantly benefit biomedical research and disease diagnostics.

A label-free colorimetric isothermal cascade amplification for the detection of disease-related nucleic acids based on double-hairpin molecular beacon.

K-Ras mutations at codon 12 play an important role in an early step of carcinogenesis. Here, a label-free colorimetric isothermal cascade amplification for ultrasensitive and specific detection of K-Ras point mutation is developed based on a double-hairpin molecular beacon (DHMB). The biosensor consists of DHMB probe and a primer-incorporated polymerization template (PPT) designed partly complementary to DHMB. In the presence of polymerase, target DNA is designed to trigger strand displacement amplification (SDA) via promote the hybridization of PPT with DHMB and subsequently initiates cascade amplification process with the help of the nicking endonuclease. During the hybridization and enzymatic reaction, G-quadruplex/hemin DNAzymes are generated, catalyzing the oxidation of ABTS2- by H2O2 in the presence of hemin. Utilizing the proposed facile colorimetric scheme, the target DNA can be quantified down to 4 pM with the dynamic response range of 5 orders of magnitude, indicating the substantially improved detection capability. Even more strikingly, point mutation in K-ras gene can be readily observed by the naked eye without the need for the labeling or expensive equipment. Given the high-performance for K-Ras analysis, the enhanced signal transduction capability associated with double-hairpin structure of DHMB provides a novel rout to screen biomarkers, and the descripted colorimetric biosensor seems to hold great promise for diagnostic applications of genetic diseases.