Effects of plasma-derived exosomes from the normal and thin Bactrian camels on hepatocellular carcinoma and their differences at transcriptome and proteomics levels.

Background: Hepatocellular carcinoma (HCC) is a common malignant primary tumor. Bactrian camels have high economic and social values, but their potential medical value has not been studied. This study aimed to investigate the effects of Bactrian camel plasma-derived exosomes on HCC. Methods: Plasma was obtained from thin and normal Bactrian camels, and used to isolate exosomes by ultracentrifugation. The exosomes were then characterized by transmission electron microscopy and Nano particle tracking analyzer. In vivo imaging of nude mice and hematoxylin eosin (HE) staining of liver tissues were used to explore the effects of the exosomes on tumor growth. Finally, the differences of the two exosomes were further analyzed using small RNA sequencing and proteomics. Results: In vivo imaging and HE staining showed that no significant differences were found in fluorescence value and liver tissue morphology between the control mice and the mice treated with the exosomes from thin Bactrian camels; while the fluorescence value and the live histology changes were alleviated in the mice with the exosomes from normal Bactrian camels. After sequencing and proteomic analysis, 40 differentially expressed miRNAs (DE-miRNAs, 15 down-regulated and 25 up-regulated) and 172 differentially expressed proteins (DEPs, 77 up-regulated and 95 down-regulated) were identified in the plasma-derived exosomes from normal Bactrian camels. These identified DE-miRNAs and DEPs were significantly enriched in many signaling pathways. Conclusions: Normal Bactrian camel plasma-derived exosomes may inhibit the growth of HCC cells through regulating pathways of Ras, Ras-Association Proximate 1 (Rap1), phosphoinositide 3-kinase-protein kinase B (PI3K-Akt), mitogen-activated protein kinase (MAPK), adenosine monophosphate-activated protein kinase (AMPK), and canonical Wnt signaling pathways.

[Changes in the Expression of AKT and ERK after the JSRV-Env-Induced Transfection of TC-1 and TC-1-Hyal2 Cells].

This study aims to explore the tumorigenic mechanism of the target cells following JSRV interaction with its receptor. We transfected mouse lung epithelial cells (TC-1) and mouse lung epithelial cells stably expressing sheep Hyal-2(TC-1-Hyal2)with JSRV-Env eukaryotic expression vector, measured the changes in the mRNA and protein expression of AKT(serine/threonine kinase)and ERK(extracellular signal-regulated kinase)in cellular signal transduction pathways, and analyzed the role of sheep Hyal-2in JSRV-Env-induced transformation of TC-1cells.First,TC-1and TC-1-Hyal2 cells were cultured in vitro and were each divided into pEGFP-C1-env transfection group,pEGFP-C1 transfection group, and untransfected group. The expression of key enzymes was determined by PCR and Western blotting. qPCR showed that, for both cell lines, compared with untransfected cells, the expression of AKT and ERK1/2mRNA was significantly increased in the pEGFP-C1-env transfected cells(P<0.05).Western blotting showed that, relative to untransfected cells, transfection with pEGFP-C1-env significantly increased p-Akt (S473)protein expression in both cell lines(P<0.05).Moreover, p-Akt (T308)and p-Erk1/2protein expression was increased significantly in the pEGFP-C1-env transfected TC-1cells(P<0.05),and very significantly in the pEGFP-C1-env transfected TC-1-Hyal2cells(P<0.01).Cells of each type transfected with the empty vector pEGFP-C1 and the untransfected cells did not show significant differences in their mRNA and protein levels of AKT and ERK(P >0.05).Thus, the expression of JSRV-Env in the cell lines TC-1and TC-1-Hyal2 activated the cellular signal transduction pathways Ras-Raf-MAPK and PI3K-Akt.The expression of AKT and ERK was significantly increased in pEGFP-C1-env transfected TC-1and TC-1-Hyal2 cells, but a greater increase was seen in the TC-1-Hyal2 cells.We speculate that Hyal2 plays a catalytic role in JSRV-Env-induced transformation of TC-1cells.

Overexpression of BAT3 alleviates prion protein fragment PrP106-126-induced neuronal apoptosis.

BACKGROUNDS AND AIMS: Prion diseases are a group of infectious neurodegenerative diseases characterized by neuronal death and degeneration. Human leukocyte antigen-B-associated transcript 3 (BAT3) is an important apoptosis regulator. We therefore investigated the interactions between BAT3 and prion protein and the potential role of BAT3 in PrP106-126-induced apoptosis. METHODS: BAT3 and prion protein were overexpressed in Hela, Neuro2A, or primary neuronal cells by transfection with BAT3-HA or PRNP-EGFP expression plasmids and their relationship studied by immunofluorescence and Western blotting. The effect of BAT3 on PrP106-126-induced cytotoxicity and apoptosis was detected by the CCK-8 assay and terminal-deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. The expression of cytochrome c and Bcl-2 was examined by Western blotting. RESULTS: BAT3 interacted with prion protein and enhanced PrP expression. After PrP106-126 peptide treated, BAT3 was transported from the nucleus to cytoplasm, increased cell viability, and protected neurons from PrP106-126-induced apoptosis through stabilizing the level of Bcl-2 protein and inhibiting the release of cytochrome c to cytoplasm. CONCLUSIONS: Our present data showed a novel molecular mechanism of PrP106-126-induced apoptotic process regulation through the overexpression of BAT3, which may be important for the basic regulatory mechanism of neuron survival in prion diseases and associated neurodegenerative diseases in vivo.

[Cloning and sequencing analyses of the complete genome of the provirus of the Inner-Mongolia pandemic strain of the Jaagsiekte sheep retrovirus].

To investigate the kinship between the Inner Mongolia pandemic strain and representative strains of the Jaagsiekte sheep retrovirus (JSRV), total DNA from the lung tissue of a JSRV-infected sheep in Inner Mongolia was used to clone fragments of gag, pro and pol genes. The recombinant plasmid pMD-JSRV (including complete genomic sequence of the JSRV strain isolated from Inner Mongolia) was constructed by linking all the cloned fragments with long terminal repeat (LTR) and env gene fragments (cloned previous and reserved by our research team). Sequence analyses revealed that the genome was 7690 bp in length and contained several typical molecular markers for exogenous form of JSRV. These included the Sca I restriction site in the gag gene, two predicted "CCHC" motifs of zinc finger in the encoded nucleocapsid protein and the predicted "YXXM" motif in the TM region of Env. Homology analyses showed that the virus strain belonged to the JSRV type II. pMD-JSRV and AF105220 strains shared a nucleotide identification of 95%. The full length genomic clone of JSRV could provide a molecular basis for an infectious JSRV molecular clone as well as an experimental platform to study the detection and pathogenesis of JSRV.

Regulation of T lymphocyte subpopulations in specific pathogen free chickens following experimental fowl adenovirus Ⅷ infection

Two-day-old specific pathogen-free (SPF) chickens were divided into two groups. Group I was inoculated orally with fowl adenovirus VIII (FAV-VIII). Group II served as a negative control. Chickens were investigated at various days post-inoculation (dpi) by flow cytometric analysis for changes in T lymphocyte subpopulations in immune system and blood. In the thymus, CD3+ T lymphocytes were increased at 25 dpi, with significant increases in the FAV infected noted at 1, 12, 20dpi (p<0.05). This was accompanied by a corresponding increase of CD4+ and CD8+ T lymphocytes. In the spleen, CD3+ and CD4+ T lymphocytes were increased significantly at 30 dpi (p<0.01) whereas CD8+ and TCR γ δ+ T lymphocytes were decreased at 1 (p<0.05), 30 dpi (p<0.01). An increase of CD3+, CD4+ and CD8+ T lymphocytes was noticed in peripheral blood, and accompanied by a decrease of TCR γ δ+ T lymphocytes. These results demonstrated that infection with FAV-VIII causes significant fluctuations in T lymphocyte subpopulations in thymus, blood and spleen. It can be concluded that an infection with FAV-VIII has profound effects on the immune system, especially on cell mediated immune competency.

Regulation of T lymphocyte subpopulations in specific pathogen-free chickens following experimental fowl adenovirus-â §VIII infection.

Two-day-old specific pathogen-free (SPF) chickens were divided into two groups. Group I was inoculated orally with fowl adenovirus Ⅷ (FAV-Ⅷ). Group II served as a negative control. Chickens were investigated at various days post-inoculation (dpi) by flow cytometric analysis for changes in T lymphocyte subpopulations in immune system and blood. In the thymus, CD3(+)T lymphocytes were increased at 25 dpi, with significant increases in the FAV infected noted at 1, 12, 20dpi (p<0.05). This was accompanied by a corresponding increase of CD4(+) and CD8(+) T lymphocytes. In the spleen, CD3(+) and CD4(+) T lymphocytes were increased significantly at 30 dpi (p<0.01) whereas CD8(+) and TCR γ δ(+) T lymphocytes were decreased at 1 (p<0.05), 30 dpi (p<0.01). An increase of CD3(+), CD4(+) and CD8(+) T lymphocytes was noticed in peripheral blood, and accompanied by a decrease of TCR γ δ(+) T lymphocytes. These results demonstrated that infection with FAV-Ⅷ causes significant fluctuations in T lymphocyte subpopulations in thymus, blood and spleen. It can be concluded that an infection with FAV-Ⅷ has profound effects on the immune system, especially on cell mediated immune competency.