Oxygenated phosphatidylethanolamine navigates phagocytosis of ferroptotic cells by interacting with TLR2.

During cancer therapy, phagocytic clearance of dead cells plays a vital role in immune homeostasis. The nonapoptotic form of cell death, ferroptosis, exhibits extraordinary potential in tumor treatment. However, the phagocytosis mechanism that regulates the engulfment of ferroptotic cells remains unclear. Here, we establish a novel pathway for phagocytic clearance of ferroptotic cells that is different from canonical mechanisms by using diverse ferroptosis models evoked by GPX4 dysfunction/deficiency. We identified the oxidized phospholipid, 1-steaoryl-2-15-HpETE-sn-glycero-3-phosphatidylethanolamine (SAPE-OOH), as a key eat-me signal on the ferroptotic cell surface. Enriching the plasma membrane with SAPE-OOH increased the efficiency of phagocytosis of ferroptotic cells by macrophage, a process that was suppressed by lipoprotein-associated phospholipase A2. Ligand fishing, lipid blotting, and cellular thermal shift assay screened and identified TLR2 as a membrane receptor that directly recognized SAPE-OOH, which was further confirmed by TLR2 inhibitors and gene silencing studies. A mouse mammary tumor model of ferroptosis verified SAPE-OOH and TLR2 as critical players in the clearance of ferroptotic cells in vivo. Taken together, this work demonstrates that SAPE-OOH on ferroptotic cell surface acts as an eat-me signal and navigates phagocytosis by targeting TLR2 on macrophages.

Predator stress-induced depression is associated with inhibition of hippocampal neurogenesis in adult male mice.

Stress has been suggested to disturb the 5-hydroxytryptamine system and decrease neurogenesis, which contribute to the development of depression. Few studies have investigated the effect of predator stress, a type of psychological stress, on depression and hippocampal neurogenesis in adult mice; we therefore investigated this in the present study. A total of 35 adult male Kunming mice were allocated to a cat stress group, cat odor stress group, cat stress + fluoxetine group, cat odor stress + fluoxetine group, or a control group (no stress/treatment). After 12 days of cat stress or cat odor stress, behavioral correlates of depression were measured using the open field test, elevated plus maze test, and dark-avoidance test. The concentrations of hippocampal 5-hydroxytryptamine and 5-hydroxyindoleacetic acid were measured using high-performance liquid chromatography-electrochemical detection. Neurogenesis was also analyzed using a bromodeoxyuridine and doublecortin double-immunostaining method. Cat stress and cat odor stress induced depression-like behaviors; this effect was stronger in the cat stress model. Furthermore, compared with the control group, cat stress mice exhibited lower 5-hydroxytryptamine concentrations, higher 5-hydroxyindoleacetic acid concentrations, and significantly fewer bromodeoxyuridine+/doublecortin+-labeled cells in the dentate gyrus, which was indicative of less neurogenesis. The changes observed in the cat stress group were not seen in the cat stress + fluoxetine group, which suggests that the effects of predator stress on depression and neurogenesis were reversed by fluoxetine. Taken together, our results indicate that depression-like behaviors induced by predator stress are associated with the inhibition of hippocampal neurogenesis.

Functionalization of Halloysite Nanotubes via Grafting of Dendrimer for Efficient Intracellular Delivery of siRNA.

Here, polyamidoamine grafted halloysite nanotubes (PAMAM- g-HNTs) were synthesized for loading of siRNA in order to intracellular delivery of siRNA and treat of breast cancer via gene therapy. The successful grafting of PAMAM on HNTs was confirmed by various analytical methods. The size, zeta potential, and grafting ratio of PAMAM- g-HNTs is ∼206.2 nm, +19.8 mV, and 3.04%, respectively. PAMAM- g-HNTs showed good cytocompatibility toward HUVECs (84.7%) and MCF-7 cells (82.3%) even at high concentration of 100 µg/mL. PAMAM- g-HNTs/siRNA exhibited enhanced cellular uptake efficiency of 94.3% compared with Lipofectamine 2000 (Lipo2000)/siRNA (83.6%). PAMAM- g-HNTs/small interfering RNA-vascular endothelial growth factor (siVEGF) led to 78.0% knockdown of cellular VEGF mRNA and induced 33.6% apoptosis in the MCF-7 cells, which is also much higher than that of Lipo2000/siVEGF. In vivo anti-cancer results demonstrated that PAMAM- g-HNTs/siVEGF treated 4T1-bearing mice showed enhanced anti-cancer efficacy than Lipo2000/siVEGF group. Also, the nanocarrier system showed negligible toxic effects toward the major organs of mice. In vivo fluorescence imaging studies showed that there is a slight decrease in the fluorescence signal of PAMAM- g-HNTs/cy5-siVEGF after 72 h post-injection. Therefore, PAMAM- g-HNTs show promising application as novel nanovectors for siRNA delivery and gene therapy of cancer.

In vitro and in vivo toxicity evaluation of halloysite nanotubes.

Because of their outstanding properties, increasing numbers of research studies and emerging applications for manufacturing products are currently in progress for halloysite nanotubes (HNTs). Therefore, the impact of HNTs on the environment and human health should be taken into consideration. In order to clearly show the cell uptake of HNTs and the biodistribution of HNTs in zebrafish, HNTs are labeled with fluorescein isothiocyanate (FITC-HNTs). The cytotoxicity assays showed that the cell viabilities of human umbilical vein endothelial cells (HUVECs) and human breast adenocarcinoma (MCF-7) cells were above 60% after being treated with different concentrations of HNTs (2.5-200 µg mL-1) for 72 h. Confocal laser scanning microscopy (CLSM) results showed the uptake of HNTs by HUVECs and MCF-7 cells. The in vivo toxicity of HNTs was then investigated in the early development of zebrafish embryos. The percent survival of zebrafish embryos and larvae showed no significant changes at different developmental stages (24, 48, 72, 96, and 120 hpf) when treated with various concentrations of HNTs (0.25-10 mg mL-1). Besides, HNTs could promote the hatchability of zebrafish embryos and did not affect the morphological development of zebrafish at a concentration of ≤25 mg mL-1. HNTs could also be ingested by zebrafish larvae and accumulated predominantly in the gastrointestinal tract. The fluorescence intensity of FITC-HNTs decreased gradually with time, which suggested that HNTs could be excreted by zebrafish larvae through the gastrointestinal metabolism. Therefore, it can be concluded that HNTs are relatively biocompatible nanomaterials, which can be utilized in many fields.

[Research on spermatozoa-mediated gene transfer to product transgenic animals].

The recombinant plasmids pLNCXHLF fused with human lactoferritin gene were directly injected into male rabbit's testis and male goat's spermaductus. The transfected males were fertilized with females one month later.Using F(1)/R(1) PCR system and southern blotting,the transgenic positive rate of rabbit offsprings genomic DNA was 35%(11/31). From the PCR results of F(1)/R(1) and F(2)/R(2) system,the transgenic positive rate of genomic DNA of goat offsprings were 33.3% (4/12) and 25% (3/12) respectively. We prepared genomic DNA from 11 kinds of tissues of goat offsprings,which were tongue, lung,liver, muscle, skin,brain, gonad, spleen, intestines, heart, and kidney. The result of F(1)/R(1) PCR system indicated that the abilities to uptake exogenous DNA were various in different tissues; the positive signals of F(2)/R(2) PCR system were feebler than the ones of F(1)/R(1) PCR system,and the density of positive signals attributed to the amount of copies of exogenous DNA in the tissues. In this experiment,spermatozoa-mediated gene transfer can produce the transgenic animals after exogenous DNA being entrapped by liposome. But during the course of fertilization and the early process of embryo proliferation,the exogenous DNA had lost segments,partly integrated,or existed outside of genomic DNA. So the rate of chimera was relatively high. According to this result,this method is not only a simple and effective way to produce transgenic animals but also make references to other researchers to prepare transgenic mammals by this means.

[Studies of the clone and cell expression of human lactoferritin gene].

In this paper,we directly cloned 2.366 Kb cDNA sequence of human lactoferrin gene and 800 bp 5'flank regulatory sequence of beta/lactoglobulin gene from goat by PCR,then connected them with the expression vector pLNCX. The recombinant plasmid pLNCXHLF containing human lactoferrin gene cDNA was transfected into mice mammary tumor cell line MA/782 after liposome transinfection. Positive single clone cells were selected with G418 and by PCR. After proliferating,the transfected cells immobilized and cultured in sodium alginate were induced by hormone. The result of Western blotting analysis on cultured cell supernatant shows that transfected cells can express the exogenic gene and secrete hLF protein, whose MW is 34 KD. The highest amount detected by ELISA reached 65 mg/l medium/10(5) cells. The result of antibacterial experiment indicates that the recombinant hLF protein has the effect of inhibiting E.coli proliferation;moreover,its activity is superior to the commercial available hLF's.