Accurate Cancer Screening and Prediction of PD-L1-Guided Immunotherapy Efficacy Using Quantum Dot Nanosphere Self-Assembly and Machine Learning.

Accurate quantification of exosomal PD-L1 protein in tumors is closely linked to the response to immunotherapy, but robust methods to achieve high-precision quantitative detection of PD-L1 expression on the surface of circulating exosomes are still lacking. In this work, we developed a signal amplification approach based on aptamer recognition and DNA scaffold hybridization-triggered assembly of quantum dot nanospheres, which enables bicolor phenotyping of exosomes to accurately screen for cancers and predict PD-L1-guided immunotherapeutic effects through machine learning. Through DNA-mediated assembly, we utilized two aptamers for simultaneous ultrasensitive detection of exosomal antigens, which have synergistic roles in tumor diagnosis and treatment prediction, and thus, we achieved better sample classification and prediction through machine-learning algorithms. With a drop of blood, we can distinguish between different cancer patients and healthy individuals and predict the outcome of immunotherapy. This approach provides valuable insights into the development of personalized diagnostics and precision medicine.

Electrochemiluminescence-Based Single-Particle Tracking of the Biomolecules Moving along Intercellular Membrane Nanotubes between Live Cells.

Electrochemiluminescence (ECL) imaging, a rapidly evolving technology, has attracted significant attention in the field of cellular imaging. However, its primary limitation lies in its inability to analyze the motion behaviors of individual particles in live cellular environments. In this study, we leveraged the exceptional ECL properties of quantum dots (QDs) and the excellent electrochemical properties of carbon dots (CDs) to develop a high-brightness ECL nanoprobe (CDs-QDs) for real-time ECL imaging between living cells. This nanoprobe has excellent signal-to-noise ratio imaging capabilities for the single-particle tracking (SPT) of biomolecules. Our finding elucidated the enhanced ECL mechanism of CDs-QDs in the presence of reactive oxygen species through photoluminescence, electrochemistry, and ECL techniques. We further tracked the movement of single particles on membrane nanotubes between live cells and confirmed that the ECL-based SPT technique using CD-QD nanoparticles is an effective approach for monitoring the transport behaviors of biomolecules on membrane nanotubes between live cells. This opens a promising avenue for the advancement of ECL-based single-particle detection and the dynamic quantitative imaging of biomolecules.

In Situ Quantitative Imaging of Plasma Membrane Stiffness in Live Cells Using a Genetically Encoded FRET Sensor.

Cell membrane stiffness is critical for cellular function, with cholesterol and sphingomyelin as pivot contributors. Current methods for measuring membrane stiffness are often invasive, ex situ, and slow in process, prompting the need for innovative techniques. Here, we present a fluorescence resonance energy transfer (FRET)-based protein sensor designed to address these challenges. The sensor consists of two fluorescent units targeting sphingomyelin and cholesterol, connected by a linker that responds to the proximity of these lipids. In rigid membranes, cholesterol and sphingomyelin are in close proximity, leading to an increased FRET signal. We utilized this sensor in combination with confocal microscopy to explore changes in plasma membrane stiffness under various conditions, including differences in osmotic pressure, the presence of reactive oxygen species (ROS) and variations in substrate stiffness. Furthermore, we explored the impact of SARS-CoV-2 on membrane stiffness and the distribution of ACE2 after attachment to the cell membrane. This tool offers substantial potential for future investigations in the field of mechanobiology.

Ratiometric Fluorescence Probes for In Situ Imaging of Membrane Tension in Live Cells.

Membrane tension is an important physical parameter of describing cellular homeostasis, and it is widely used in the study of cellular processes involving membrane deformation and reorganization, such as cell migration, cell spreading, and cell division. Despite the importance of membrane tension, direct measurement remains difficult. In this work, we developed a ratiometric fluorescent probe sensitive to membrane tension by adjusting the carbon chain structure based on polarity-sensitive fluorophores. The probe is sensitive to changes in membrane tension after cells were subjected to physical or chemical stimuli, such as osmotic shock, lipid peroxidation, and mechanical stress. When the polarity of the plasma membrane increases (the green/red ratio decreases) and the membrane tension increases, the relative magnitude of the membrane tension can be quantitatively calculated by fluorescence ratio imaging. Thus, the probe proved to be an efficient and sensitive membrane tension probe.

Artificial Cells for Cellular Behavior Mimicry Induced by Sphingomyelin Degradation.

Sphingomyelinase (SMase), a hydrolase of sphingomyelin (SM) enriched in the outer leaflet of the plasma membrane of mammalian cells, is closely associated with the onset and development of many diseases, but the specific mechanisms of SMase on the cell structure, function, and behavior are not yet fully understood due to the complexity of the cell structure. Artificial cells are minimal biological systems constructed from various molecular components designed to mimic cellular processes, behaviors, and structures, which are excellent models for studying biochemical reactions and dynamic changes in cell membranes. In this work, we presented an artificial cell model that mimics the lipid composition and content of the outer leaflet of mammalian plasma membranes for studying the effect of SMase on cell behavior. The results confirmed that the artificial cells can respond to SM degradation by producing ceramides that enrich and alter the membrane charge and permeability, thus inducing the budding and fission of the artificial cells. Thus, the artificial cells developed here provide a powerful tool to study the mechanism of action of cell membrane lipids on cell biological behavior, paving the way for further molecular mechanism studies.

Engineered Lipidic Nanomaterials Inspired by Sphingomyelin Metabolism for Cancer Therapy.

Sphingomyelin (SM) and its metabolites are crucial regulators of tumor cell growth, differentiation, senescence, and programmed cell death. With the rise in lipid-based nanomaterials, engineered lipidic nanomaterials inspired by SM metabolism, corresponding lipid targeting, and signaling activation have made fascinating advances in cancer therapeutic processes. In this review, we first described the specific pathways of SM metabolism and the roles of their associated bioactive molecules in mediating cell survival or death. We next summarized the advantages and specific applications of SM metabolism-based lipidic nanomaterials in specific cancer therapies. Finally, we discussed the challenges and perspectives of this emerging and promising SM metabolism-based nanomaterials research area.

ADP-Ribosylation Factor Like GTPase 4C (ARL4C) augments stem-like traits of glioblastoma cells by upregulating ALDH1A3.

Glioma cells with stem cell-like properties are crucial for tumor initiation, progression and therapeutic resistance. Therefore, identifying specific factors in regulating stem-like traits is critical for the design of novel glioma therapeutics. Herein, we reported that ADP-Ribosylation Factor Like GTPase 4C (ARL4C) was highly expressed in glioma stem-like cells (GSLCs). GSLCs, determined by the efficiency of sphere formation in vitro and tumor growth in vivo, was increased by overexpression of ARL4C. ARL4C induced the tumorigenesis through ALDH1A3. Analyses of 325 patient specimens showed that ARL4C was highly expressed in glioblastoma (GBM) as compared with lower grade gliomas. In addition, higher level ARL4C expression in glioma was correlated with poorer progression-free survival and overall survival of patients. Therefore, ARL4C may act as a novel prognostic marker and a therapeutic target for GBM.

[Development of a universal primers PCR-coupled liquid bead array to detect biothreat bacteria].

OBJECTIVE: To develop a fast, high-throughput screening method with suspension array technique for simultaneous detection of biothreat bacteria. METHODS: 16 S rDNA universal primers for Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp.and Burkholderia pseudomallei were selected to amplify corresponding regions and the genus-specific or species-specific probes were designed. After amplification of chromosomal DNA by 16 S rDNA primers 341A and 519B, the PCR products were detected by suspension array technique. The sensitivity, specificity, reproducibility and detection power were also analyzed. RESULTS: After PCR amplification by 16 S rDNA primers and specific probe hybridization, the target microorganisms could be identified at genus level, cross reaction was recognized in the same genus. The detection sensitivity of the assay was 1.5 pg/microl (Burkholderia pseudomallei), 20 pg/microl (Brucella spp.), 7 pg/microl (Bacillus anthracis), 0.1 pg/microl (Francisella tularensis), and 1.1 pg/microl (Yersinia pestis), respectively. The coefficient of variation for 15 test of different probes was ranged from 5.18% to 17.88%, it showed good reproducibility. The assay could correctly identify Bacillus anthracis and Yersinia pestis strains in simulated white powder samples. CONCLUSION: The suspension array technique could be served as an opening screening method for biothreat bacteria rapid detection.

[Development of a multiplex PCR-suspension array for simultaneous detection of five bioterrorism bacteria].

OBJECTIVE: To develop a rapid, high-throughput screening method of gene suspension array technique to simultaneously detect five bioterrorism bacteria: Bacillus anthracis, Francisella tularensis, Yersinia pestis, Brucella spp. and Burkholderia pseudomallei. METHODS: Highly validated specific primers were used to amplify diagnostic regions unique to each pathogen. Biotin labelled PCR products were hybridized to corresponding probes coupling on the unique sets of fluorescent beads. The hybridized beads were processed through the Bio-plex, which identified the presence of PCR products. RESULTS: Multiplex PCR-suspension array can detect five bioterrorism bacteria correctly with high specificity and high sensitivity, the results suggest the utility of suspension array system for high-throughput screening of bioterrorism samples. CONCLUSION: A multiplex PCR-suspension array for rapid detection of five bioterrorism bacteria was established.

[Study on the correlation of the levels of serum IFN-γ and RANTES and rabbit vulnerable atherosclerotic plaque].

AIM: To investigate the correlation between the levels of serum IFN-γ and RANTES (regulated upon on activation on normal T cell expressed and secreted) and rabbit atherosclerotic plaque stability. METHODS: 30 male New Zealand rabbits were randomly divided into three groups: control group, stable atherosclerotic plaque group(AS group) and vulnerable atherosclerotic plaque group(VAP group), with 10 rabbits in a group. The control group was given normal diet while the AS group and the VAP group were given high-fat diet for 12 weeks. According to literature method pharmacological triggers inducing the vulnerable atherosclerotic plaque formation in the VAP group. Fasting blood was extracted from ear medium artery about 2 mL at week-0 and the 12th week, serum total cholesterol, triglyceride and high density lipoprotein cholesterol were detected by enzymatic method, and low density lipoprotein cholesterol was calculated by Friedwald formula. All animals were executed after they were fed by injected excessive Phenobarbital sodium at ear vein for 12 weeks. Before executed, ear medium artery blood was taken to detect the levels of serum IFN-γ and RANTES by ELISA.The corrected plaque area and vulnerability index were calculated by Masson staining and its correlation coefficient with inflammatory factors were measured. RESULTS: The level of serum IFN-γ at the AS group and the VAP group, compared with the control group, was significantly increased(all P<0.01), at the same time, the level of serum IFN-γ in the VAP group was significantly higher than in the AS group(P<0.01). About the level of serum RANTES there was no obvious difference when the AS group was compared with the control group(P>0.05). The level of serum RANTES in the VAP group was higher than in the AS group and the control group(all P<0.01). Regarding corrected plaque area and vulnerability Index, the VAP group was significantly higher than the AS group (P<0.01). In the AS group and the VAP group, the serum concentrations of IFN-γand RANTES exhibited a positive correlation with corrected plaque area and vulnerability Index (P<0.05). CONCLUSION: IFN-γ and RANTES may be inflammatory marker about atherosclerotic vulnerable plaque, and their serum concentration can be used to evaluate the atherosclerotic plaque instability.

[Nifedipine regulated expression of bcl-2 in human gingival epithelial cells in vitro].

PURPOSE: To investigate the effect of nifedipine(NIF) on transcription of bcl-2 in human gingival epithelial cells(HGECs) in vitro and to study the pathogenesis of epithelial thickening in drug-induced gingival overgrowth(DGO). METHODS: The gingival tissues obtained from periodontal surgeries were digested with enzyme and HGECs were cultured in vitro; HGECs were identified by immunohistochemistry; bcl-2 mRNA levels were quantitated by Real-time PCR 24 hours and 48 hours after cells stimulated by NIF with different concentration (0microg/ml, 1microg/ml,2microg/ml,3microg/ml), in which 0microg/ml NIF as blank control. The data was analyzed by one-way ANOVA using SPSS 11.0 software package. RESULTS: HGECs cultured in vitro showed keratin positive signal and vimentin negative signal; the level of bcl-2 mRNA increased with NIF 3microg/ml after 24 hours treatment, which appeared significant increase compared with blank control (P<0.05); after 48 hours treatment the level of bcl-2 mRNA in the groups of 2microg/ml and 3microg/ml showed significant increase compared with blank control (P<0.05). CONCLUSION: NIF regulates the level of bcl-2 mRNA.

[Amelogenin expression in cultured human dental pulp and periodontium related cells].

PURPOSE: To investigate gene expression of amelogenin (Am) in human gingival epithelial cells(HGEC) and also other oral ectomesenchyme cells (human gingival fibroblasts, human periodontal ligament fibroblasts and human pulp cells). METHODS: The amelogenin mRNA expression patterns were determined by the reverse transcription polymerase chain reaction (RT-PCR),and the protein expression was studied with Western blotting. RESULTS: There was no amelogenin expression detected in any of the cells. CONCLUSIONS: These findings suggest that amelogenin expression could not be detected in cultured human periodontium-related cells. Supported by National Natural Science Foundation of China (Grant No.30672315) and Research Fund of Science and Technology Commission of Shanghai Municipality(Grant No.08DZ2271100).