Single-cell analysis defines highly specific leukemia-induced neutrophils and links MMP8 expression to recruitment of tumor associated neutrophils during FGFR1 driven leukemogenesis.

BACKGROUND: Leukemias driven by activated, chimeric FGFR1 kinases typically progress to AML which have poor prognosis. Mouse models of this syndrome allow detailed analysis of cellular and molecular changes occurring during leukemogenesis. We have used these models to determine the effects of leukemia development on the immune cell composition in the leukemia microenvironment during leukemia development and progression. METHODS: Single cell RNA sequencing (scRNA-Seq) was used to characterize leukemia associated neutrophils and define gene expression changes in these cells during leukemia progression. RESULTS: scRNA-Seq revealed six distinct subgroups of neutrophils based on their specific differential gene expression. In response to leukemia development, there is a dramatic increase in only two of the neutrophil subgroups. These two subgroups show specific gene expression signatures consistent with neutrophil precursors which give rise to immature polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Analysis of gene expression in these precursor cells identified pathways that were specifically upregulated, the most pronounced of which involved matrix metalloproteinases Mmp8 and Mmp9, during leukemia progression. Pharmacological inhibition of MMPs using Ilomastat preferentially restricted in vitro migration of neutrophils from leukemic mice and led to a significantly improved survival in vivo, accompanied by impaired PMN-MDSC recruitment. As a result, levels of T-cells were proportionally increased. In clinically annotated TCGA databases, MMP8 was shown to act as an independent indicator for poor prognosis and correlated with higher neutrophil infiltration and poor pan-cancer prognosis. CONCLUSION: We have defined specific leukemia responsive neutrophil subgroups based on their unique gene expression profile, which appear to be the precursors of neutrophils specifically associated with leukemia progression. An important event during development of these neutrophils is upregulation MMP genes which facilitated mobilization of these precursors from the BM in response to cancer progression, suggesting a possible therapeutic approach to suppress the development of immune tolerance.

Green tea polyphenol epigallocatechin-3-gallate ameliorates insulin resistance in non-alcoholic fatty liver disease mice.

AIM: Epigallocatechin-3-gallate (EGCG) is a major polyphenol in green tea. In this study, we investigated the effects of EGCG on insulin resistance and insulin clearance in non-alcoholic fatty liver disease (NAFLD) mice. METHODS: Mice were fed on a high-fat diet for 24 weeks. During the last 4 weeks, the mice were injected with EGCG (10, 20 and 40 mg·kg(-1)·d(-1), ip). Glucose tolerance, insulin tolerance and insulin clearance were assessed. After the mice were euthanized, blood samples and tissue specimens were collected. Glucose-stimulated insulin secretion was examined in isolated pancreatic islets. The progression of NAFLD was evaluated histologically and by measuring lipid contents. Insulin-degrading enzyme (IDE) protein expression and enzyme activity were detected using Western blot and immunocapture activity assays, respectively. RESULTS: The high-fat diet significantly increased the body weight and induced grade 2 or 3 liver fatty degeneration (steatosis, lobular inflammation and ballooning) accompanied by severe hyperlipidemia, hyperglycemia, hyperinsulinemia and insulin resistance in the model mice. Administration of EGCG dose-dependently ameliorated the hepatic morphology and function, reduced the body weight, and alleviated hyperlipidemia, hyperglycemia, hyperinsulinemia and insulin resistance in NAFLD mice. Furthermore, EGCG dose-dependently enhanced insulin clearance and upregulated IDE protein expression and enzyme activity in the liver of NAFLD mice. CONCLUSION: EGCG dose-dependently improves insulin resistance in NAFLD mice not only by reducing body weight but also through enhancing the insulin clearance by hepatic IDE. The results suggest that IDE be a potential drug target for the treatment of NAFLD.

[Resveratrol protects against hydrogen peroxide induced injury in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effect of Resveratrol on Hydrogen peroxide-treated human umbilical vein endothelial cells (HUVEC). METHODS: Oxidative stress in HUVEC was induced by hydrogen peroxide (H2O2). The cells were divided in to 5 groups: A. Control group; B. H2O2 group; C. Res + H2O2 group; D. Res + H2O2 + LY294002 group; E. H2O2 + LY294002 group. The cell viability were measured by cck-8 assay; Malondialdehyde (MDA) and Superoxide dismutase (SOD) were determined by using commercially available kits; Reactive oxygen species (ROS)were detect by flow cytometer; the expression of Nuclear factor-E2-related factor(Nrf2), Protein kinase B(Akt), Mammalian target of rapamycin (mTOR) were determined by Western blot. RESULTS: Compared with control, resveratrol increased the cells viability (36.14 ± 2.20) to (72.05 ± 2.46) (P = 0.004), decreased MDA formation (299.38 ± 12.42) to (159.17 ± 7.30)(P = 0.006), elevated SOD activity(35.61 ± 3.26) to (81.37 ± 4.55) (P = 0.007), inhibited ROS production (177.86 ± 8.98) to (133.96 ± 4.17) (P = 0.003). These changes were accompany with the activation of p-Nrf2 (198.57 ± 6.89) to (371.10 ± 8.41) (P = 0.005), p-Akt (102.93 ± 3.29) to (221.62 ± 6.51) (P = 0.004), p-mTOR (93.26 ± 4.12) to (172.14 ± 5.74) (P = 0.008). However these protective effect of resveratrol were abolished by LY294002. CONCLUSION: Resvertrol could protects HUVEC cells from H2O2-induced oxidative stress, in which the PI3K-Akt-mTOR pathway may be involved.

A gold nanorods-based fluorescent biosensor for the detection of hepatitis B virus DNA based on fluorescence resonance energy transfer.

In this study, we designed a fluorescence resonance energy transfer system containing gold nanorods (AuNRs) and fluorescein (FAM) for the detection of hepatitis B virus DNA sequences. AuNRs were synthesized according to the seed-mediated surfactant-directed approach, and the surface of the AuNRs was wrapped with a thin layer of cetyltrimethylammonium bromide (CTAB), resulting in the AuNRs being positively charged. When FAM-tagged single-stranded DNA (FAM-ssDNA) was added into the AuNRs suspension, it was adsorbed onto the surface of the positively charged AuNRs and formed a FAM-ssDNA-CTAB-AuNRs ternary complex, the resulting structure led to a fluorescence resonance energy transfer (FRET) process from FAM to AuNRs and the fluorescence intensity of FAM was consequently quenched. When complementary target DNA was added to the FAM-ssDNA-CTAB-AuNRs complex solution, a further decrease in fluorescence intensity was observed because of an increased FRET efficiency. Under optimal conditions, the decline of the fluorescence intensity of FAM (ΔF) was linear with the concentration of the complementary DNA from 0.045 to 6.0 nmol L(-1) and the detection limit was as low as 15 pmol L(-1) (signal/noise ratio of 3). When this fluorescent DNA sensor was used to detect the polymerase chain reaction product of hepatitis B virus gene extracted from a positive real sample, a positive response was obtained. Impressively, the biosensor exhibits good selectivity, even for single-mismatched DNA detection.

Anti-inflammatory effect of soyasaponins through suppressing nitric oxide production in LPS-stimulated RAW 264.7 cells by attenuation of NF-κB-mediated nitric oxide synthase expression.

The anti-inflammatory properties of soyasaponins (especially soyasaponins with different chemical structures) have scarcely been investigated. We investigated the inhibitory effects of five structural types of soyasaponins (soyasaponin A(1), A(2), I and soyasapogenol A, B) on the induction of nitric oxide (NO) and inducible NO synthase (iNOS) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS). Soyasaponin A(1), A(2) and I (25-200 µg/mL) dose-dependently inhibited the production of NO and tumor necrosis factor α (TNF-α) in LPS-activated macrophages, whereas soyasapogenol A and B did not. Furthermore, soyasaponin A(1), A(2) and I suppressed the iNOS enzyme activity and down-regulated the iNOS mRNA expression both in a dose-dependent manner. The reporter gene assay revealed that soyasaponin A(1), A(2) and I decreased LPS-induced nuclear factor kappa B (NF-κB) activity. Soyasaponin A(1), A(2) and I exhibit anti-inflammatory properties by suppressing NO production in LPS-stimulated RAW 264.7 cells through attenuation of NF-κB-mediated iNOS expression. It is proposed that the sugar chains present in the structures of soyasaponins are important for their anti-inflammatory activities. These results have important implication for using selected soyasaponins towards the development of effective chemopreventive and anti-inflammatory agents.