BACKGROUND:miRNAs expression has been reported to be associated with hepatic and renal fibrosis,and dermal fibrogenesis. Moreover,a targeted regulatory relationship between miR-192-5p and epidermal regulators has been demonstrated in gouty arthritis.OBJECTIVE:To investigate the expression and regulatory role of miR-192-5p in hypertrophic scar and to verify whether there is a targeted regulatory relationship between miR-192-5p and epidermal regulators. METHODS:(1) Six cases of hypertrophic scar tissue and six cases of normal skin tissue were collected from the First Affiliated Hospital of Xinjiang Medical University. And miR-192-5p and epidermal regulator mRNA expression were detected by qRT-PCR. (2) The primary hypertrophic scar fibroblasts were obtained using tissue explant method and cultured to 3-6 generations for subsequent experiments. There were three groups in the experiment:negative control group,miR-192-5p mimic group and miR-192-5p inhibitor group. The latter two groups were transfected with the corresponding sequences. Cell proliferation viability was detected by the cell counting kit-8 assay and EdU kit;and the migration ability was detected by the cell scratch test. Cell apoptosis was detected by flow cytometry. The gene and protein expressions of epidermal regulator,type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were detected by qRT-PCR and western blot,respectively. miR-192-5p targets were predicted by a bioinformatics website,and target binding was validated by dual luciferase assay. RESULTS AND CONCLUSION:(1) Compared with normal skin tissues and their fibroblasts,miR-192-5p and epidermal regulator were highly expressed in hypertrophic scar and hypertrophic scar fibroblasts (P<0.05 or P<0.01). (2) After overexpression of miR-192-5p,cell proliferation was enhanced (P<0.05) and EdU positive cell rate increased (P<0.01) when compared with the negative control group;after inhibition of miR-192-5p,cell viability (P<0.05) and EdU positive rate decreased (P<0.05). (3) At 24 hours after overexpression of miR-192-5p,compared with the negative control group,the area between cell scratches and apoptosis rate decreased in the miR-192-5p mimic group (P<0.05) but increased in the miR-192-5p inhibitor group (P<0.01). (4) At 48 hours after transfection,the mRNA and protein levels of epidermal regulator were significantly decreased in the miR-192-5p mimic group,while the mRNA and protein levels of type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were significantly increased (P<0.05 or P<0.01). The miR-192-5p inhibitor group showed opposite changes in the above four indicators (P<0.05 or P<0.01). (5) The Targetscan website predicted that epidermal regulator had a potential binding site for miR-192-5p. (6) Dual luciferase assays showed that miR-192-5p could bind to epidermal regulator in a targeted manner. To conclude,overexpression of miR-192-5p can decrease the expression of epidermal regulator,and the two may be negatively regulated,suggesting that regulation of epidermal regulator may play a role in inhibiting the proliferation of hypertrophic scar fibroblasts.

BACKGROUND:miRNAs expression has been reported to be associated with hepatic and renal fibrosis,and dermal fibrogenesis. Moreover,a targeted regulatory relationship between miR-192-5p and epidermal regulators has been demonstrated in gouty arthritis.OBJECTIVE:To investigate the expression and regulatory role of miR-192-5p in hypertrophic scar and to verify whether there is a targeted regulatory relationship between miR-192-5p and epidermal regulators. METHODS:(1) Six cases of hypertrophic scar tissue and six cases of normal skin tissue were collected from the First Affiliated Hospital of Xinjiang Medical University. And miR-192-5p and epidermal regulator mRNA expression were detected by qRT-PCR. (2) The primary hypertrophic scar fibroblasts were obtained using tissue explant method and cultured to 3-6 generations for subsequent experiments. There were three groups in the experiment:negative control group,miR-192-5p mimic group and miR-192-5p inhibitor group. The latter two groups were transfected with the corresponding sequences. Cell proliferation viability was detected by the cell counting kit-8 assay and EdU kit;and the migration ability was detected by the cell scratch test. Cell apoptosis was detected by flow cytometry. The gene and protein expressions of epidermal regulator,type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were detected by qRT-PCR and western blot,respectively. miR-192-5p targets were predicted by a bioinformatics website,and target binding was validated by dual luciferase assay. RESULTS AND CONCLUSION:(1) Compared with normal skin tissues and their fibroblasts,miR-192-5p and epidermal regulator were highly expressed in hypertrophic scar and hypertrophic scar fibroblasts (P<0.05 or P<0.01). (2) After overexpression of miR-192-5p,cell proliferation was enhanced (P<0.05) and EdU positive cell rate increased (P<0.01) when compared with the negative control group;after inhibition of miR-192-5p,cell viability (P<0.05) and EdU positive rate decreased (P<0.05). (3) At 24 hours after overexpression of miR-192-5p,compared with the negative control group,the area between cell scratches and apoptosis rate decreased in the miR-192-5p mimic group (P<0.05) but increased in the miR-192-5p inhibitor group (P<0.01). (4) At 48 hours after transfection,the mRNA and protein levels of epidermal regulator were significantly decreased in the miR-192-5p mimic group,while the mRNA and protein levels of type Ⅰ collagen,type Ⅲ collagen and α-smooth muscle actin were significantly increased (P<0.05 or P<0.01). The miR-192-5p inhibitor group showed opposite changes in the above four indicators (P<0.05 or P<0.01). (5) The Targetscan website predicted that epidermal regulator had a potential binding site for miR-192-5p. (6) Dual luciferase assays showed that miR-192-5p could bind to epidermal regulator in a targeted manner. To conclude,overexpression of miR-192-5p can decrease the expression of epidermal regulator,and the two may be negatively regulated,suggesting that regulation of epidermal regulator may play a role in inhibiting the proliferation of hypertrophic scar fibroblasts.

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

To construct a recombinant fowl adenovirus 4(FAdV-4)expressing the Cap protein of goose astrovirus genotype 2(GoAstV-2),the expression cassette of Cap gene was inserted into the natural 1 966 bp deletion region of the FAdV-4 genome in the infectious clone p15A-cm-FAdV4-HNJZ.The resulted recombinant plasmid p15A-cm-FAdV4-HNJZ-Cap/GoAstV-2 was linearized with restriction enzyme and transfected into chicken hepatoma cell line(LMH)to rescue the recombinant FAdV-4 expressing the Cap protein of GoAstV-2,rF Ad V4-Cap/GoAstV-2.After 15 passages in LMH cells,the recombinant rFAdV4-Cap/GoAstV-2 was identified by PCR using primers flanking the insertion site of the Cap gene expression cassette and using viral genome DNA extracted from rFAdV4-Cap/GoAstV-2 infected LMH cells as template.LMH cells were in-fected with 15th passage rFAdV4-Cap/GoAstV-2 and indirect immunofluorescence was performed with a polyclonal antibody against Cap protein as the primary antibody.Western blot was carried out with lysates of rFAdV4-Cap/GoAstV-2 infected LMH cells.The in vitro replication dynamic of the 15th passage of the rFAdV4-Cap/GoAstV-2 was also investigated in LMH cells.The results demonstrated that the Cap gene of GoAstV-2 was presented in the genome of the recombinant vi-rus rF AdV4-Cap/Go Ast V-2,and could be expressed stably.The prepared recombinant virus in this study will lay a foundation for developing inactivated bivalent vaccine candidate against co-in-fection of FAdV-4 and GoAstV-2 in goose.

Ethyl carbamate (EC) is a carcinogenic substance in many fermented foods. Enzymatic removal of ethyl carbamate from fermented foods is an important way to eliminate its potential health damage to consumers. To study the enzymatic properties of an ethyl carbamate hydrolase (urethanase) from Klebsiella pneumoniae, a strain isolated from murine somach, we purified the enzyme using ammonium sulfate precipitation, ion exchange chromatography and gel filtration chromatography. The molecular mass of this enzyme was estimated to be 55 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Its K(m) was 74 mmol/L when EC was used as the substrate. Moreover, its optimal reaction temperature was 55 degrees C, and the optimum pH was 7.0. The activity was enhanced by ethylene diamine tetraacetic acid (EDTA) and dithiothreitol (DTT), but strongly inhibited by Cu2+ and Zn2+. The enzyme was halophilic and tolerant to low concentration of ethanol. Therefore, it has the potential to remove EC from fermented foods.
Amidohydrolases ; chemistry ; isolation & purification ; Bacterial Proteins ; chemistry ; isolation & purification ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Hydrogen-Ion Concentration ; Klebsiella pneumoniae ; enzymology ; Molecular Weight ; Substrate Specificity ; Temperature