KLRG1-expressing CD8+ T cells are exhausted and polyfunctional in patients with chronic hepatitis B.

Killer cell lectin-like receptor G1 (KLRG1) has traditionally been regarded as an inhibitory receptor of T cell exhaustion in chronic infection and inflammation. However, its exact role in hepatitis B virus (HBV) infection remains elusive. CD8+ T cells from 190 patients with chronic hepatitis B were analyzed ex vivo for checkpoint and apoptosis markers, transcription factors, cytokines and subtypes in 190 patients with chronic hepatitis B. KLRG1+ and KLRG1- CD8+ T cells were sorted for transcriptome analysis. The impact of the KLRG1-E-cadherin pathway on the suppression of HBV replication mediated by virus-specific T cells was validated in vitro. As expected, HBV-specific CD8+ T cells expressed higher levels of KLRG1 and showed an exhausted molecular phenotype and function. However, despite being enriched for the inhibitory molecules, thymocyte selection-associated high mobility group box protein (TOX), eomesodermin (EOMES), and Helios, CD8+ T cells expressing KLRG1 produced significant levels of tumour necrosis factor (TNF)-α, interferon (IFN)-γ, perforin, and granzyme B, demonstrating not exhausted but active function. Consistent with the in vitro phenotypic assay results, RNA sequencing (RNA-seq) data showed that signature effector T cell and exhausted T cell genes were enriched in KLRG1+ CD8+ T cells. Furthermore, in vitro testing confirmed that KLRG1-E-cadherin binding inhibits the antiviral efficacy of HBV-specific CD8+ T cells. Based on these findings, we concluded that KLRG1+ CD8+ T cells are not only a terminally exhausted subgroup but also exhibit functional diversity, despite inhibitory signs in HBV infection.

A novel combined therapeutic strategy of Nano-EN-IR@Lip mediated photothermal therapy and stem cell inhibition for gastric cancer.

Recurrence and metastasis of gastric cancer is a major therapeutic challenge for treatment. The presence of cancer stem cells (CSCs) is a major obstacle to the success of current cancer therapy, often leading to treatment resistance and tumor recurrence and metastasis. Therefore, it is important to develop effective strategies to eradicate CSCs. In this study, we developed a combined therapeutic strategy of photothermal therapy (PTT) and gastric cancer stem cells (GCSCs) inhibition by successfully synthesizing nanoliposomes loaded with IR780 (photosensitizer) and EN4 (c-Myc inhibitor). The nanocomposites are biocompatible and exhibit superior photoacoustic (PA) imaging properties. Under laser irradiation, IR780-mediated PTT effectively and rapidly killed tumor cells, while EN4 synergistically inhibited the self-renewal and stemness of GCSCs by suppressing the expression and activity of the pluripotent transcription factor c-Myc, preventing the tumor progression of gastric cancer. This Nano-EN-IR@Lip is expected to be a novel clinical nanomedicine for the integration of gastric cancer diagnosis, treatment and prevention.

Immunomultiple PCR-based electrochemical and lateral flow strategy for the simultaneous detection of liver cancer tumor markers.

The current use of the single serum biomarker α-fetoprotein (AFP) in clinical practice has limitations in terms of specificity and sensitivity. We propose a strategy that combines antigen capture polymerase chain reaction (AC-PCR), lateral flow assay (LFA), and electrochemical biosensors to detect both AFP and circulating tumor cells (CTCs) in liver cancer serum. First, we used the AC-PCR technique to achieve target separation, purification, signal conversion, and amplification, eliminating target heterogeneity. Then, we achieved rapid results through the LFA and electrochemical biosensor platforms. As a result, the proposed assay has limits of 5 cells/mL for CTCs and 5 µg/L for AFP. The proposed method was applied effectively to simulated blood samples. This method has the potential to play a role in early liver cancer and provide a potential application for the diagnosis and precision treatment of liver cancer.

Preparation and Properties of Polyvinylpyrrolidone/Sodium Carboxymethyl Cellulose Soluble Microneedles.

Transdermal drug delivery is a new means of delivering drugs through the skin to achieve therapeutic effects. Microneedles have several advantages, including low cost, easy self-administration, and high delivery efficiency. Different polymers affect the morphology, mechanical properties, and drug delivery efficiency of microneedles. To study the performance and limitations of microneedles (MNs), we prepared different ratios of polymers. MNs were fabricated from polyvinylpyrrolidone (PVP) and sodium carboxymethyl cellulose (CMC-Na) using the centrifugal molding method. Needle morphology, formability, and other properties of the polymers were evaluated to compare the performances of MNs with different ratios. PVP and CMC-Na were intermixed at different ratios with water as the solvent. The soluble MNs were prepared by mold casting. The morphology, thermodynamic properties, and crystallinity were studied using scanning electron microscopy (SEM), thermogravimetric analysis (TG), differential scanning calorimetric analysis (DSC), and X-ray diffraction (XRD). The results showed that composite microneedles have good thermal stability. Among the different compositions tested, the 10% PVP/2% CMC-Na composite microneedle demonstrated the best performance with a regular surface morphology and relatively high thermal decomposition and melting temperatures. These results indicate that microneedles with appropriate ratios of two different materials possess good formability and other properties.

XNA probe and CRISPR/Cas12a-powered flexible fluorescent and electrochemical dual-mode biosensor for sensitive detection of m6A site-specific RNA modification.

N6-methyladenosine (m6A) in RNAs is closely related to various biological progresses, but the specific regulatory mechanisms are still unclear. The existing m6A single-base resolution analysis techniques have problems of specificity and sensitivity to be improved, which can hardly meet the urgent needs of basic research and clinical applications. This work proposes a new strategy based on xeno nucleic acid (XNA) probe and CRISPR/Cas12a signal amplification for the sensitive detection of site-specific m6A modifications. According to the difference in the thermodynamic stability of hybridization between XNA probe with m6A-RNA and A-RNA, XNA was designed as a block probe to mediate m6A-RNA specific reverse transcription polymerase chain reaction (MsRT-PCR). Therefore, m6A can be specifically distinguished by converting difficult-to-test m6A modifications into easily detectable dsDNA fragments. Integration of CRISPR/Cas12a technology, skilfully designed sequences of crRNAs targeting m6A site-specific amplification dsDNA. The specificity was significantly improved through dual specific recognition of XNA probe and crRNA. Furthermore, the sensitivity of the assay was also greatly increased by the combined signal amplification of PCR and CRISPR/Cas12a. Additionally, we extend the application of CRISPR/Cas12a to flexible fluorescent and electrochemical biosensing system, which can accurately detect m6A modifications with different ranges of methylation fractions. The analysis results of m6A sites in MALAT1, ACTB and TPT1 further demonstrated the feasibility of the constructed biosensor for the accurate detection of hypomethylated samples in cells. The implementation of this work will provide strong technical support to promote the in-depth research on m6A in disease regulation mechanisms and in vitro molecular diagnosis.

Analysis of glycan expression on cell surfaces by using a glassy carbon electrode modified with MnO2 nanosheets and DNA-generated electrochemical current.

Electrochemical assay for analysis of cell surface glycan expression is reported. Mannose on human breast cancer cells (type MCF-7) is selected as the glycan model. Gold nanoparticles are modified with binding aptamer for MCF-7 cells and act as electrochemical probe. The analysis of cell surface glycan expression follows a traditional sandwich protocol. Concanavalin A that can specifically recognize mannose is immobilized onto MnO2 nanosheets modified electrode for the capture of MCF-7 cells. Then, the modified gold nanoparticles are immobilized onto the electrode via the binding between MCF-7 cell and aptamer on the gold nanoparticles. The aptamer on the gold nanoparticles reacts with molybdate. More specifically, the reaction of the phosphate backbone of aptamer with molybdate results in the formation of a redox-active molybdophosphate precipitate and generates an electrochemical current. The current intensity at 0.20 V (vs. Ag/AgCl) is recorded to test the linear range of the assay. The assay shows an obvious response to MCF-7 cells with a wide linear range from 1.0 × 103 to 1.0 × 106 cells mL-1 and a limit of detection down to 300 cells mL-1. The assay can be used to selectively monitor the change of mannose expression on cell surfaces upon the treatment with the N-glycan inhibitor. Graphical abstractSchematic of an electrochemical assay for analysis of cell surface glycan expression of MCF-7 cancer cells.

Enhanced photocatalytic performance of magnetic multi-walled carbon nanotubes/cerium dioxide nanocomposite.

An easily separated photocatalyst, magnetic multi-walled carbon nanotubes/cerium dioxide (MMWCNTs-CeO2) nanocomposite, has been successfully prepared by hydrothermal synthesis and subsequent loading of magnetic nanoparticles. The synthesized nanocomposite was characterized by scanning electron microscope, transmission electron microscopy, X-ray photoelectron spectroscopy, powder X-ray diffraction, and vibrating sample magnetometer. The characterization results indicated the successful synthesis of MMWCNTs-CeO2 nanocomposite. Photocatalytic degradation experiments were conducted to evaluate photocatalytic properties of MMWCNTs-CeO2 by using methylene blue (MB) as a model pollutant. After a light irradiation of two hours, relatively high degradation efficiency (97.5%) of MB was achieved by using MMWCNTs-CeO2 nanocomposite as photocatalyst in the presence of H2O2. The incorporation of magnetic nanoparticles could not only facilitate the separation of photocatalyst from the solution after treatment, but also enhance the photocatalytic degradation efficiency. The results of this study suggested that the synthesized MMWCNTs-CeO2 nanocomposite showed an attractive prospect for application in the treatment of organic wastewater.