Multi-omics Integrated Analysis of the Protective Effect of EZH2 inhibition in Mice with Renal Ischemia-Reperfusion Injury

Introduction: Acute kidney injury (AKI) is a common clinical syndrome associated with high morbidity and mortality. Inhibition of the methyltransferase Enhancer of Zeste Homolog 2 (EZH2) by its inhibitor 3-DZNeP (3-Deazaneplanocin A) exerts renal benefits in acute renal ischemia-reperfusion injury (IRI). However, the underlying mechanisms is not completely known. This study aimed to elucidate the pathological mechanism of EZH2 in renal IRI by combination of multi-omics analysis and expression profiling in a public clinical cohort. Methods: In this study, C57BL/6J mice were used to establish acute kidney injury model, which were treated with 3-DZNeP for 24 hours. Kidney samples were collected for RNA-seq analysis, which was combined with publicly available EZH2-ChIP-seq data of mouse embryonic stem cell for a joint analysis to identify differentially expressed genes. Several selected differentially expressed genes were verified by quantitative PCR. Finally, single-nucleus sequencing data and expression profiling in public clinical datasets were used to confirm the negative correlation of the selected genes with EZH2 expression. Results: 3-DZNeP treatment significantly improved renal pathology and function in IRI mice. Through RNA-seq analysis combined with EZH2 ChIP-seq database, 162 differentially expressed genes were found, which might be involved in EZH2-mediated pathology in IRI kidneys. Four differential expressed genes (Scd1, Cidea, Ghr, and Kl) related to lipid metabolism or cell growth were selected based on GO and KEGG enrichment analysis, which were validated by quantitative PCR. Data from snRNA-seq revealed the negative correlation of these four genes with Ezh2 expression in different subpopulations of proximal tubular cells in IRI mice in different pattern. Finally, the negative correlation of these four genes with EZH2 expression was confirmed in patients with acute kidney injury in two clinical datasets. Conclusions: Our study indicates that Scd1, Cidea, Ghr, and Kl are downstream genes regulated by EZH2 in AKI. Upregulation of EZH2 in AKI inhibits the expression of these four genes in different population of proximal tubular cells to minimize normal physiological function, and promote acute or chronic cell injuries following acute kidney injury

Dynamic Response of UHMW-PE Composite Armors under Ballistic Impact of Blunt Projectiles

To study the dynamic response of UHMW-PE composite armor under ballistic impact, two kinds of UHMW-PE composite armors are designed. Both of them are composed of UHMW-PE laminates and steel face sheets of Q235. The blunt projectile is made of 35CrMnSiA, with a cylinder shape. By numerical simulation, the dynamic response and deformation of composite armors are obtained under the penetration of the projectile. With the increase of impact velocity, the penetration depth increases nearly linearly, with a more severe tendency of swaging in the projectile. Then, experiments are carried out to validate the numerical simulation results. Based on a ballistic gun with a caliber of 14.5 mm, the projectiles are fired with a velocity from 680 m/s to 1300 m/s. The penetration into the composite armor can be divided into an initial shear plugging stage and the following bulging and delamination stage. Based on the theoretical analysis, the shear strength in the shear plugging stage can be estimated. Associated with typical experimental results, numerical simulation is suitable to predict the bulging characteristics of the composite armor. The failure mode of the composite armors under the impact of blunt projectiles is determined, and the failure mechanism is analyzed. The penetration results in the experiment agree well with the numerical simulation results, which validate the correctness of the numerical simulation models. The research results can be significant in the design of composite armor with UHMW-PE laminates

The long non-coding RNA MALAT1 is increased in renal ischemia-reperfusion injury and inhibits hypoxia-induced inflammation

Background: To investigate the expression of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in renal ischemia-reperfusion injury and explore its role in acute kidney injury. Methods: 18 mice were randomly divided into a sham operation group (Sham) and an ischemia-reperfusion group (IR) in which animals were sacrificed at 6 h or 12 h after surgery. The kidneys were harvested to measure the expression of MALAT1 mRNA. HK2 cells were treated with cobalt chloride (CoCl2) to mimic hypoxia or transfected with siRNA to knockdown MALAT1 before CoCl2 treatment. After that, MALAT1 was analyzed by RT-PCR (reverse transcription-polymerase chain reaction). HIF-1ɑ (hypoxia-inducible factor-1 alpha) and NF-κB (nuclear factor-kappa B) was measured by Western blot. The concentrations of IL-6 (interleukin-6) and TNF-ɑ (tumor necrosis factor-alpha) in the media were detected by ELISA (enzyme-linked immunosorbent assay). Results: The expression of MALAT1 in the IR (6 h/12 h) group was significantly higher than that in the sham group. In HK2 cells, MALAT1 was significantly increased at 1 h, 3 h, and 6 h after CoCl2 treatment but had reduced to the basal level at 12 h and 24 h. Knockdown of MALAT1 by siRNA significantly up-regulated the expression of HIF-1ɑ and NF-κB proteins in CoCl2-treated HK2 cells. In addition, the concentrations of IL-6 and TNF-ɑ were increased by MALAT1 siRNA transfection in CoCl2-treated HK2 cells. Conclusion: The expression of MALAT1 is increased in renal ischemia-reperfusion injury. It is likely that MALAT1 inhibits the hypoxia-induced inflammatory response through the NF-κB pathway

Potential-Resolved Electrochemiluminescence for Simultaneous Determination of Triple Latent Tuberculosis Infection Markers

A novel electrochemiluminescence (ECL) immunosensor based on the potential-resolved strategy was first developed for simultaneous determination of triple latent tuberculosis infection (LTBI) markers with high sensitivity, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and interleukin (IL)-2. In this work, luminol and self-prepared carbon quantum dots and CdS quantum dots were integrated onto gold nanoparticles and magnetic beads in sequence to fabricate potential-resolved ECL nanoprobes with signal amplification. IFN-γ-antibody (Ab)­1, TNF-α-Ab1, and IL-2-Ab1 were separately immobilized on three spatially resolved areas of a patterned indium tin oxide electrode to capture the corresponding LTBI markers, which were further recognized by IFN-γ-Ab2, TNF-α-Ab2, and IL-2-Ab2-functionalized ECL nanoprobes. The binding reaction of antibody-functionalized ECL nanoprobes and the captured LTBI markers will generate three sensitive and potential-resolved ECL signals during one potential scanning, and the ECL intensities reflect the concentrations of IFN-γ, TNF-α, and IL-2 in the range of 1.6–200 pg mL^–1. Therefore, the multiplexed ECL immunosensor provided an effective approach for simultaneous detection of triple LTBI markers in human serum, so it will be beneficial to facilitate more accurate and reliable clinical diagnosis for LTBI

Novel Electrochemiluminescence-Sensing Platform for the Precise Analysis of Multiple Latent Tuberculosis Infection Markers

Latent tuberculosis infection (LTBI) is one of the major contributing factors for the high incidence of tuberculosis, and the low contents of LTBI markers in human serum present a great challenge for the diagnosis of LTBI. Here, we reported a novel electrochemiluminescence (ECL)-sensing platform for the precise analysis of multiple LTBI markers, interferon-gamma (IFN-γ) and interleukin (IL)-2. In this approach, self-prepared carbon quantum dots (CQDs) and luminol were integrated onto gold nanoparticles (AuNPs), which were further enriched on the surface of magnetic bead (MB) to create two solid-phase ECL nanoprobes (MB@Au@CQDs and MB@Au@luminol) for improving the detection sensitivity efficiently. Graphene oxide (GO) and AuNPs were electrodeposited onto a patterned indium tin oxide (ITO) electrode with two spatially resolved areas in sequence to form two sensitive and stable sensing areas. IFN-γ-antibody (Ab)₁ and IL-2-Ab₁ were separately immobilized on the two sensing areas to capture the corresponding LTBI markers, which were further recognized by IFN-γ-Ab₂ and IL-2-Ab₂ labeled as MB@Au@CQDs and MB@Au@luminol. The ECL intensity depended linearly on the content of IFN-γ and IL-2 in the range of 0.01–1000 pg mL^–1, with a low detection limit of 10 fg mL^–1. The proposed ECL-sensing platform is simple, sensitive, accurate, reliable, and specific to the detection of rare IFN-γ and IL-2 in human serum and provides a valuable protocol for facilitating fast and precise diagnosis of LTBI

Dual Electrochemiluminescence Signal System for <i>In Situ</i> and Simultaneous Evaluation of Multiple Cell-Surface Receptors

A mutiplex cytosensor based on a dual electrochemiluminescence (ECL) signal system was fabricated for <i>in situ</i> and simultaneous detection of the expression levels of multiple cell-surface receptors, mannose and epidermal growth factor receptor (EGFR), using luminol-capped gold nanoparticles (Au@luminol) and CdS quantum dots (CdS QDs) as potential-resolved ECL nanoprobes. Two spatially resolved areas on indium tin oxide (ITO) electrodes were modified with polyaniline (PANI) by electropolymerization, on which gold nanoparticles (AuNPs) were attached to strengthen conductivity and stability of the sensing interface. Human mucin1 protein (MUC1) aptamer was immobilized onto AuNPs for capturing MUC1-positive MCF-7 cells. Au@luminol and CdS QDs as ECL nanoprobes were covalently linked with concanavalin A (ConA) and epidermal growth factor (EGF) to label MCF-7 cells on the two areas of the cytosensor separately. Compared to conventional multiplex biosensor, we demonstrated a novel analysis platform for the simultaneous detection of multiple cell-surface receptors; it could provide two sensitive and potential-resolved ECL signals during one potential scanning and avoid cross-reactivity between the two nanoprobes. The quantification of MCF-7 cells on the two spatially resolved areas could be achieved over the linear range from 10² to 1.0 × 10⁶ cells mL^–1 with a detection limit of 20 cells mL^–1. This multiplex cytosensor was further applied for simultaneous quantitative evaluation of the expression levels of mannose and EGFR on MCF-7 cells, revealed that the average numbers of mannose and EGFR per captured MCF-7 cell were 1.2 × 10⁶ and 0.86 × 10⁵ with the relative standard deviation of 5.3% and 4.2%, respectively. The multiplex cytosensor was capable of evaluating multiple cell-surface receptors, which would be beneficial to developing a better diagnostic tool for diseases

Novel Single-Cell Analysis Platform Based on a Solid-State Zinc-Coadsorbed Carbon Quantum Dots Electrochemiluminescence Probe for the Evaluation of CD44 Expression on Breast Cancer Cells

A novel single-cell analysis platform was fabricated using solid-state zinc-coadsorbed carbon quantum dot (ZnCQDs) nanocomposites as an electrochemiluminescence (ECL) probe for the detection of breast cancer cells and evaluation of the CD44 expression level. Solid-state ZnCQDs nanocomposite probes were constructed through the attachment of ZnCQDs to gold nanoparticles and then the loading of magnetic beads to amplify the ECL signal, exhibiting a remarkable 120-fold enhancement of the ECL intensity. Hyaluronic acid (HA)-functionalized solid-state probes were used to label a single breast cancer cell by the specific recognition of HA with CD44 on the cell surface, revealing more stable, sensitive, and effective tagging in comparison with the water-soluble CQDs. This strategy exhibited a good analytical performance for the analysis of MDA-MB-231 and MCF-7 single cells with linear range from 1 to 18 and from 1 to 12 cells, respectively. Furthermore, this single-cell analysis platform was used for evaluation of the CD44 expression level of these two cell lines, in which the MDA-MB-231 cells revealed a 2.8–5.2-fold higher CD44 expression level. A total of 20 single cells were analyzed individually, and the distributions of the ECL intensity revealed larger variations, indicating the high cellular heterogeneity of the CD44 expression level on the same cell line. The as-proposed single-cell analysis platform might provide a novel protocol to effectively study the individual cellular function and cellular heterogeneity