Crosstalk between 5-methylcytosine and N6-methyladenosine machinery defines disease progression, therapeutic response and pharmacogenomic landscape in hepatocellular carcinoma.

BACKGROUND: Accumulated evidence highlights the significance of the crosstalk between epigenetic and epitranscriptomic mechanisms, notably 5-methylcytosine (5mC) and N6-methyladenosine (m6A). Herein, we conducted a widespread analysis regarding the crosstalk between 5mC and m6A regulators in hepatocellular carcinoma (HCC). METHODS: Pan-cancer genomic analysis of the crosstalk between 5mC and m6A regulators was presented at transcriptomic, genomic, epigenetic, and other multi-omics levels. Hub 5mC and m6A regulators were summarized to define an epigenetic and epitranscriptomic module eigengene (EME), which reflected both the pre- and post-transcriptional modifications. RESULTS: 5mC and m6A regulators interacted with one another at the multi-omic levels across pan-cancer, including HCC. The EME scoring system enabled to greatly optimize risk stratification and accurately predict HCC patients' clinical outcomes and progression. Additionally, the EME accurately predicted the responses to mainstream therapies (TACE and sorafenib) and immunotherapy as well as hyper-progression. In vitro, 5mC and m6A regulators cooperatively weakened apoptosis and facilitated proliferation, DNA damage repair, G2/M arrest, migration, invasion and epithelial-to-mesenchymal transition (EMT) in HCC cells. The EME scoring system was remarkably linked to potential extrinsic and intrinsic immune escape mechanisms, and the high EME might contribute to a reduced copy number gain/loss frequency. Finally, we determined potential therapeutic compounds and druggable targets (TUBB1 and P2RY4) for HCC patients with high EME. CONCLUSIONS: Our findings suggest that HCC may result from a unique synergistic combination of 5mC-epigenetic mechanism mixed with m6A-epitranscriptomic mechanism, and their crosstalk defines therapeutic response and pharmacogenomic landscape.

Multiplexed Immunoassay Using Quantum Dots to Monitor Proteins Secreted from Single Cells at Near-Single Molecule Resolution.

Single-cell secretion studies find important applications in molecular diagnostics, therapeutic target identification, and basic biology research. One increasingly important area of research is non-genetic cellular heterogeneity, a phenomenon that can be studied by assessing secretion of soluble effector proteins from single cells. This is particularly impactful for immune cells, as secreted proteins such as cytokines, chemokines, and growth factors are the gold standard for identifying phenotype. Current methods that rely upon immunofluorescence suffer from low detection sensitivity, requiring thousands of molecules to be secreted per cell. We have developed a quantum dot (QD)-based single-cell secretion analysis platform that can be used in different sandwich immunoassay formats to dramatically lower detection threshold, such that only one to a few molecules need be secreted per cell. We have also expanded this work to include multiplexing capabilities for different cytokines and employed this platform to study macrophage polarization under different stimuli at the single-cell level.

Black Phosphorus as a Targeting PPAR-γ Agonist to Reverse Chemoresistance in Patient-derived Organoids, Mice, and Pancreatic Tumor Cells.

Black phosphorus (BP) exhibits significant potential for clinical applications. However, further research is necessary to uncover the unknown biological functions of BP and broaden its applications across various fields. This study investigates the potential of BP as a targeting PPAR-γ agonist to overcome chemoresistance in the treatment of pancreatic adenocarcinoma (PAAD) using 2D and 3D cell lines, patient-derived organoids (PDOs), and mouse models. RNA-sequencing analysis shows that BP treatment enriches differentially expressed genes in the PPAR pathway, and molecular modeling predicts the potential docking site between BP and PPAR-γ. Transcriptional activity assays are further to verify the activation of PPAR-γ. BP-activated PPAR-γ inhibits cancer stem cell (CSC) properties and expression of biomarkers such as CD44 and c-Myc, which are involved in chemoresistance. Notably, CD44 overexpression in tumor cells renders them susceptible to BP while insensitive to gemcitabine. This indicates that BP preferentially targets stem-like cells, which exhibit heightened resistance to chemotherapeutic drugs. A combination treatment strategy involving BP and gemcitabine is developed, demonstrating enhanced treatment efficacy of PAAD in both in vitro and in vivo models. Thus, BP serves as a PPAR-γ agonist capable of reversing chemoresistance, establishing it as a potent anti-tumor approach for the treatment of PAAD.

Macrophage-organoid co-culture model for identifying treatment strategies against macrophage-related gemcitabine resistance.

BACKGROUND: Gemcitabine resistance (GR) is a significant clinical challenge in pancreatic adenocarcinoma (PAAD) treatment. Macrophages in the tumor immune-microenvironment are closely related to GR. Uncovering the macrophage-induced GR mechanism could help devise a novel strategy to improve gemcitabine treatment outcomes in PAAD. Therefore, preclinical models accurately replicating patient tumor properties are essential for cancer research and drug development. Patient-derived organoids (PDOs) represent a promising in vitro model for investigating tumor targets, accelerating drug development, and enabling personalized treatment strategies to improve patient outcomes. METHODS: To investigate the effects of macrophage stimulation on GR, co-cultures were set up using PDOs from three PAAD patients with macrophages. To identify signaling factors between macrophages and pancreatic cancer cells (PCCs), a 97-target cytokine array and the TCGA-GTEx database were utilized. The analysis revealed CCL5 and AREG as potential candidates. The role of CCL5 in inducing GR was further investigated using clinical data and tumor sections obtained from 48 PAAD patients over three years, inhibitors, and short hairpin RNA (shRNA). Furthermore, single-cell sequencing data from the GEO database were analyzed to explore the crosstalk between PCCs and macrophages. To overcome GR, inhibitors targeting the macrophage-CCL5-Sp1-AREG feedback loop were evaluated in cell lines, PDOs, and orthotopic mouse models of pancreatic carcinoma. RESULTS: The macrophage-CCL5-Sp1-AREG feedback loop between macrophages and PCCs is responsible for GR. Macrophage-derived CCL5 activates the CCR5/AKT/Sp1/CD44 axis to confer stemness and chemoresistance to PCCs. PCC-derived AREG promotes CCL5 secretion in macrophages through the Hippo-YAP pathway. By targeting the feedback loop, mithramycin improves the outcome of gemcitabine treatment in PAAD. The results from the PDO model were corroborated with cell lines, mouse models, and clinical data. CONCLUSIONS: Our study highlights that the PDO model is a superior choice for preclinical research and precision medicine. The macrophage-CCL5-Sp1-AREG feedback loop confers stemness to PCCs to facilitate gemcitabine resistance by activating the CCR5/AKT/SP1/CD44 pathway. The combination of gemcitabine and mithramycin shows potential as a therapeutic strategy for treating PAAD in cell lines, PDOs, and mouse models.

Visual detection of copper(II) ions in blood samples by controlling the leaching of protein-capped gold nanoparticles.

We have developed a simple, low-cost, paper-based probe for the selective colorimetric detection of copper ions (Cu(2+)) in aqueous solutions. The bovine serum albumin (BSA)-modified 13.3-nm Au nanoparticle (BSA-Au NP) probe was designed to detect Cu(2+) ions using lead ions (Pb(2+)) and 2-mercaptoethanol (2-ME) as leaching agents in a glycine-NaOH (pH 12.0) solution. In addition, a nitrocellulose membrane (NCM) was used to trap the BSA-Au NPs, leading to the preparation of a nanocomposite film consisting of a BSA-Au NP-decorated membrane (BSA-Au NPs/NCM). The BSA-Au NPs probe operates on the principle that Cu deposition on the surface of the BSA-Au NPs inhibits their leaching ability, which is accelerated by Pb(2+) ions in the presence of 2-ME. Under optimal solution conditions (5 mM glycine-NaOH (pH 12.0), Pb(2+) (50 µM), and 2-ME (1.0 M)), the Pb(2+)/2-ME-BSA-Au NPs/NCM enabled the detection of Cu(2+) at nanomolar concentrations in aqueous solutions by the naked eye with high selectivity (at least 100-fold over other metal ions). In addition, this cost-effective probe allowed for the rapid and simple determination of Cu(2+) ions in not only natural water samples but also in a complex biological sample (in this case, blood sample).

The Antibacterial Effects of Supermolecular Nano-Carriers by Combination of Silver and Photodynamic Therapy.

The over-use of antibiotics has promoted multidrug resistance and decreased the efficacy of antibiotic therapy. Thus, it is still in great need to develop efficient treatment strategies to combat the bacteria infection. The antimicrobial photodynamic therapy (aPDT) and silver nanoparticles have been emerged as effective antibacterial methods. However, the silver therapy may induce serious damages to human cells at high concentrations and, the bare silver nanoparticles may rapidly aggregate, which would reduce the antibacterial efficacy. The encapsulation of sliver by nano-carrier is a promising way to avoid its aggregation and facilitates the co-delivery of drugs for combination therapy, which does not require high concentration of sliver to exert antibacterial efficacy. This work constructed a self-assembled supermolecular nano-carrier consisting of the photosensitizers (PSs), the anti-inflammatory agent and silver. The synthesized supermolecular nano-carrier produced reactive oxygen species (ROS) under the exposure of 620-nm laser. It exhibited satisfying biocompatibility in L02 cells. And, this nano-carrier showed excellent antibacterial efficacy in Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) as indicated by bacterial growth and colony formation. Its antibacterial performance is further validated by the bacteria morphology through the scanning electron microscope (SEM), showing severely damaged structures of bacteria. To summary, the supermolecular nano-carrier TCPP-MTX-Ag-NP combining the therapeutic effects of ROS and silver may serve as a novel strategy of treatment for bacterial infection.

Effects of three drying methods on polyphenol composition and antioxidant activities of Litchi chinensis Sonn.

The aim of this study was to investigate the effects of three different drying methods, freeze drying (FD), vacuum drying (VD) and oven drying (OD) on phenolic contents and antioxidant activities of litchi fruits. 20 polyphenols were exactly identified in the litchi fruits by UPLC-QqQ/MS. Significant losses were observed in the contents of total polyphenols and antioxidant activities in the dried litchi when compared with the fresh litchi. Principle component analysis indicated that there was significant difference of phenolic component between the use of thermal drying (VD and OD) and FD. Our results suggest that FD is the optimum drying method for litchi fruits considering the content of total polyphenols and antioxidant activities.