The DNA demethylase genes are widespread in plants. Four DNA demethylase genes (LJDME1, LJDME2, LJDME3 and LJDME4) were obtained from transcriptome dataset of Lonicera japonica Thunb by using bioinformatics methods and the proteins' physicochemical properties they encoded were predicted. The phylogenetic tree showed that the four DNA demethylase genes and Arabidopsis thaliana DME had a close relationship. The result of gene expression model showed that four DNA demethylase genes were different between species. The expression levels of LJDME1 and LJDME2 were even more higher in Lonicera japonica var. chinensis than those in L. japonica. LJDME] and LJDME2 maybe regulate the active compounds of L. japonica. This study aims to lay a foundation for further understanding of the function of DNA demethylase genes in L. japonica.

Objective The core mechanism of renal insterstitial fibrosis (RIF) is epithelial-mesenchymal transition.This study aimed to investigate the effect of erythropoietin on high glucose-induced epithelial-mesenchymal transition ( EMT) of normal hu-man kidney proximal tubular epithelial cells (HK-2) and its possible mechanism. Mothods HK-2 cells cultured in vitro were ran-domly divided into a blank control group , a high glucose induction group , a mannitol induction group , an EPO induction group , an EPO (5, 10, and 20U/mL) inhibition group, and an Rho kinase inhibitor group.After 24 hours of intervention, the mRNA levels of RhoA and ROCK were determined by RT-PCR, those of E-cadherin and α-smooth muscle actin (α-SMA) proteins detected by immu-nofluorescence staining , and the expression of FN proteins in the supernatant measured by ELISA . Results Compared with the blank control group , the expressions of RhoA and ROCK 1 mRNA were significantly increased in the high glucose induction group (0.945 ±0.132 vs 1.400 ±0.022, 1.007 ±0.002 vs 1.913 ±0.011, P<0.05), but markedly decreased in the 5, 10, and 20U/mL EPO inhibition groups (1.400 ±0.022 vs 1.278 ±0.006, 1.400 ±0.022 vs 0.770 ±0.005, 1.400 ±0.022 vs 0.334 ±0.009, P<0.006) in comparison with the high glucose induction group , and the effects were related to the concentration of EPO .Compared with the blank control, the expression of E-cadherin protein was increased in the high glucose induction group (0.644 ±0.006 vs 0.107 ± 0.004, P<0.05), but remarkably decreased in the 5, 10, and 20 U/mL EPO inhibition groups (0.236 ±0.006, 0.433 ±0.010, 0.521 ±0.010) in comparison with the high glucose induction group (P<0.05), and the effects were also related to the concentration of EPO.Pearson correlation analysis showed a positive correlation between the mRNA expressions of RhoA and ROCK 1 in the high glu-cose induction and EPO inhibition groups . Conclusion EPO can inhibit high glucose-induced epithelial-mesenchymal transition of normal human kidney HK-2 cells and thus delay renal fibrosis , which mignt be related to the RhoA/ROCK signaling pathway .

Objective To simulate the mechanical environment of cells in vivo and study cellular signal transduction mechanisms. Methods A device was developed which could provide high cell yield, control the time, strain magnitude, direction and frequency of stretch, and applied 10% cyclic strain to cell culture substrate with stretch frequency at 1Hz. Results After being stretched, morphology and cytoskeleton of cells were altered. The major axis of cells and the alignment of stress fibers were vertical to the orientation of cyclic stretch. Conclusion This device provides versatile options for the study on the cellular responses of mechanical loading.

OBJECTIVE: To observe the effects of matrine on proliferation and apoptosis of human renal cell carcinoma cell line GRC-1 in vitro, and to explore its mechanism. METHODS: The human renal cell carcinoma cell line GRC-1 was treated with matrine of different concentrations for 24, 48, 72 and 96 h respectively. The MTT assay was used to evaluate the cytotoxic effects of matrine on GRC-1 cells. The transmission electron microscope and flow cytometry were utilized to observe and detect the apoptosis of GRC-1 cells induced by matrine. The expression levels of Bcl-2 and Bax proteins were evaluated by streptavidin-biotin-peroxidase method. RESULTS: The matrine of different concentrations all have cytotoxic effects on GRC-1 cells, with obvious dose- and time-dependent effects. The apoptosis induced by matrine was confirmed in GRC-1 cells. With intervention of matrine (1.5 g/L) for 12 h, the expression level of Bcl-2 in GRC-1 cells was decreased while the expression level of Bax was increased as compared with those in the untreated group. CONCLUSION: The proliferation-inhibiting effects of matrine on human renal cell carcinoma cell line GRC-1 may be related to down-regulating the ratio of Bcl-2/Bax protein expression and promoting the apoptosis.

In the face of escalating problems with pathogen control, the development of proper formulations of existing antibiotics is as important as the development of novel antibiotics. Daptomycin is a lipopeptide antibiotic with potent activity against Gram-positive bacteria. Currently, only injectable solution of daptomycin has been approved for clinical use. In the present study, the formulation of PEGylated liposomal daptomycin (PLD) was prepared and optimized, and its efficacy against methicillin-resistant Staphylococcus aureus (MRSA252) strains was investigated. The obtained PLD had a mean vesicle diameter of (111.5 +/- 15.4) nm and a mean percent drug loading of (5.81 +/- 0.19) % with high storage stability. Potent activity of PLD against MRSA was demonstrated in vitro with a more sustained effect than that of conventional liposomal daptomycin and daptomycin solution. In addition, intravenous administration of a single dose (equal to human use) of PLD significantly increased the survival of mice in a MRSA252 systemic infection model compared with other formulations. Drug distribution in the lung was significantly enhanced following administration of PLD, and no measurable tissue lesions or pathological changes were detected during PLD treatment. Taken together, PEGylated liposomes loaded with daptomycin may represent a promising approach to reduce MRSA252 infections, especially those involving bloodstream dissemination, such as hematogenous pulmonary infection.
Animals ; Anti-Bacterial Agents ; pharmacology ; Daptomycin ; pharmacology ; Liposomes ; Methicillin-Resistant Staphylococcus aureus ; drug effects ; Mice ; Staphylococcal Infections ; drug therapy

The DNA demethylase genes are widespread in plants. Four DNA demethylase genes (LJDME1, LJDME2, LJDME3 and LJDME4) were obtained from transcriptome dataset of Lonicera japonica Thunb by using bioinformatics methods and the proteins' physicochemical properties they encoded were predicted. The phylogenetic tree showed that the four DNA demethylase genes and Arabidopsis thaliana DME had a close relationship. The result of gene expression model showed that four DNA demethylase genes were different between species. The expression levels of LJDME1 and LJDME2 were even more higher in Lonicera japonica var. chinensis than those in L. japonica. LJDME] and LJDME2 maybe regulate the active compounds of L. japonica. This study aims to lay a foundation for further understanding of the function of DNA demethylase genes in L. japonica.
Computational Biology ; DNA, Plant ; chemistry ; Genes, Plant ; Lonicera ; enzymology ; genetics ; Oxidoreductases, O-Demethylating ; genetics ; Phylogeny ; Plant Proteins ; genetics ; Transcriptome

Objective To study the effects of erythropoietin (rhEPO) in high glucose induced proliferation and apopto?sis of human kidney proximal tubular epithelial (HK-2) cells, and the possible mechanism thereof. Methods HK-2 cells cultured in vitro were divided into several groups randomly:blank control group, high glucose group, mannitol group, rhEPO control group, different concentrations of rhEPO treatment groups (5, 10, 20 U/mL) and Rho kinase group. The reverse tran?scription polymerase chain reaction (RT-PCR) was used to evaluate the mRNA levels of RhoA and ROCK after 24 hours. Tetrazolium salt method (MTT) was used to determine the cell proliferation. Cell apoptosis was detected by flow cytometry. Results Compared with blank control group the expression levels of RhoA and ROCK1 mRNA were significantly in?creased in high glucose group (P < 0.05). RhoA, ROCK1 mRNA expressions significantly decreased in rhEPO group than those of high glucose group (P<0.05). There was a positive correlation between the expression levels of RhoA mRNA and ROCK1 mRNA in high glucose group and rhEPO group. MTT method showed that rhEPO significantly promoted the prolifer?ation of HK-2 cells (P<0.05). Flow cytometry analysis showed that high glucose induced apoptosis in HK-2 cells, which was significantly inhibited in rhEPO group and Rho kinase group as compared to that of high glucose group in a concentra?tion dependent manner (P<0.05). Conclusion rhEPO can promote HK-2 cell proliferation and inhibit apoptosis, which may be related to RhoA/ROCK signaling pathway.

Objective To investigate the frequencies of circulating dendritic cell (DC) subsets and the function of monocyte-derived dendritic cells in patients with hepatitis B-related acute-on-chronic liver failure.Methods Peripheral blood was collected from hepatitis B-related acute-on-chronic liver failure patients (ACLF,n =40) and chronic hepatitis B (CHB,n =40) as well as normal controls (NCs,n =20).Circulating myeloid dendritic cell (Mdc) and plasmic dendritic cell (pDC) frequencies in peripheral blood mononuclear cells (PBMC) were analyzed by flow cytometric analysis.Purified monocytes were isolated by combination of Histopaque-1.077 and CD14 Microbeads.Monocyte-derived dendritic cells (MoDCs) generated in vitro in the presence of interleukin (IL)-4 and granulocyte macrophage colony-stimulating factor upon activation by poly I:C.Costimulatory molecule expression and allostimulatory mixed lymphocyte reaction (AMLR) of MoDCs were detected in patients with hepatitis B-related ACLF.Results The number of circulating mDC decreased only in patients with hepatitis B-related ACLF compared with that in normal controls.However,pDC numbers decreased in both CHB and ACLF patients.We observed a further decrease the pDC numbers in ACLF compared to CHB patients without statistical significance (P ＞ 0.05).MoDC from ACLF patients showed lower expression of costimulatory molecules CD80,CD86 and the mature marker CD83,as well as MHC Ⅱ molecule (HLA-DR) compared to CHB and NC group.Interestingly,MoDC impaired allostimulatory mixed lymphocyte reaction from ACLF patients compared to those in CHB patients and NCs.Conclusions Patients with hepatitis B-related ACLF have a significantly lower expression of surface markers and impaired AMLR of MoDC,as well as decreased number of circulating mDC and pDC,which may be partially related to HBV disease progression in these patients.

BACKGROUND: Tumor necrosis factor-α (TNF-α) is one of important cytokines to promote the maturation of dendritic cells. Blockage of TNF-α action by binding with soluble tumor necrosis factor receptor 1 (sTNFR1) may arrest dendritic cells in an immature state and induce stable, long-term tolerance. OBJECTIVE: To construct the lentiviral vectors carrying sTNFR1 gene and investigate sTNFR1 expression in immature dendritic cells. METHODS: Total RNA of human peripheral blood mononuclear cells was taken as a template. The sTNFR1 gene fragment was amplified by RT-PCR, subcloned to the lentiviral vectors pXZ208, and ligated to the enhanced green fluorescent protein (eGFP) reporter gene to establish lentiviral vector, called pXZ9-sTNFR1. DNA sequencing was performed for lentiviral vector identification. Lentivirus was prepared by transfection of 293 FT cells with pXZ9-sTNFR1. Viral titer was determined by eGFP expression. C57BL/6 mouse bone marrow-derived dendritic cells were in vitro cultured with low-dose granulocyte-macrophage colony stimulating factors and interleukin 4. On day 5 of culture, immature dendritic cells were transfected with pXZ9-sTNFR1 recombinant lentiviral supernatant, sTNFR1 transcription was detected by RT-PCR, sTNFR1 protein expression by Western blot analysis. Following sTNFR1 gene modification and lipopolysaccharide stimulation, the phenotype characteristics of dendritic cells were observed. RESULTS AND CONCLUSION: Recombinant plasmid pXZ9-sTNFR1 was successfully constructed. Twenty-four hours after 293 FT cell transfection, eGFP expression was observed and viral titer was over 10<'6> U/L. RT-PCR demonstrated that pXZ9-sTNFRl-transfected immature dendritic cells showed sTNFR1 positive expression. Western blot analysis revealed that sTNFR1 protein appeared in the immature dendritic cells and supernatant following 293 FT cell transfection. On day 5 of culture, dendritic cells expressed low level of class Ⅱ major histocompatibility complex (MHC Ⅱ), as well as CD40, CD86, CD80, molecules. However, following lipopolysaccharide stimulation, dendritic cells expressed high level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules, exhibiting the phenotype characteristics of mature dendritic cells. But after sTNFR modification, the expression level of MHC Ⅱ, as well as CD40, CD80, and CD86, molecules was not altered obviously. Lentiviral vectors carrying sTNFR1 gene and eGFP reporter gene were successfully constructed, and recombinant lentiviral plasmids with high titer were acquired. Following high efficacy of lentiviral gene transfection, immature dendritic cells stably express sTNFR1 mRNA and protein, which prevents immature dendritic cells from activation by exogenous lipopolysaccharide and maintains the immature state.

BACKGROUND: Cells in contact with nanomaterials can induce oxidative stress, allergic reactions, and then produce cytotoxicity and genotoxicity. Therefore, studies on nano toxicology have attracted more and more attention.OBJECTIVE: To comparatively evaluate the cytocompatibility of Pluronic (P85, F127, F87) tri-block copolymer nanoparticles modified with folic acid (FA) and polylactic acid (PLA).METHODS: Pluronic (P85, F127, F87) tri-block copolymer nanoparticles were modified with FA and PLA to synthesize a variety of amphiphilic block copolymers, including PLA-P85-PLA, FA-P85-PLA, PLA-F127-PLA, FA-F127-PLA,PLA-F87-PLA and FA-F87-PLA. The cytotoxicity of these synthesized nanoparticles was analyzed by cell morphology,cell metabolic activity and cell membrane effects in HepG-2 cells.RESULTS AND CONCLUSION: The relative growth rate of HepG-2 cells had no significant differences under 24-hour induction of various concentrations (5, 10, 20, 50, 100 mg/L) of unmodified P85, F127, and F87 nanoparticles (P > 0.05).The growth and proliferation of cells under the low concentrations (5, 10, 20, 50 mg/L) were enhanced. P85 NPs and F87 NPs could significantly inhibit cell viability at dose of 400 mg/L. In contrast, there were no significant differences towards P85, F127 and F87 nanoparticles (5, 10, 20, 50, 100, 200, 400 mg/L) modified with FA and PLA when compared with the control group (P > 0.05). These findings indicate that the modification of FA and PLA can improve the cytocompatibility of Pluronic (P85, F127, F87) tri-block copolymers, and therefore, PLA-Pluronic-PLA and FA-Pluronic-PLA nanoparticles are both good candidates for drug vectors.