CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozy...CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.展开更多
AIM:To find a possible relationship between inflammation and CA19-9 tumor marker by analyzing data from patients with benign jaundice(BJ) and malignant jaundice(MJ).METHODS:All patients admitted for obstructive jaundi...AIM:To find a possible relationship between inflammation and CA19-9 tumor marker by analyzing data from patients with benign jaundice(BJ) and malignant jaundice(MJ).METHODS:All patients admitted for obstructive jaundice,in the period 2005-2009,were prospectively enrolled in the study,obtaining a total of 102 patients.On admission,all patients underwent complete standard blood test examinations including C-reactive protein(CRP),bilirubin,CA19-9.Patients were considered eligible for the study when they presented obstructive jaundice confirmed by instrumental examinations and increased serum bilirubin levels(total bilirubin > 2.0 mg/dL).The standard cut-off level for CA19-9 was 32 U/mL,whereas for CRP this was 1.5 mg/L.The CA19-9 level was adjusted by dividing it by the value of serum bilirubin or by the CRP value.The patients were divided into 2 groups,MJ and BJ,and after the adjustment a comparison between the 2 groups of patients was performed.Sensitivity,specificity and positive predictive values were calculated before and after the adjustment.RESULTS:Of the 102 patients,51 were affected by BJ and 51 by MJ.Pathologic CA19-9 levels were found in 71.7% of the patients.In the group of 51 BJ patients there were 29(56.9%) males and 22(43.1%) females with a median age of 66 years(range 24-96 years),whereas in the MJ group there were 24(47%) males and 27(53%) females,with a mean age of 70 years(range 30-92 years).Pathologic CA19-9 serum level was found in 82.3% of MJ.CRP levels were pathologic in 66.6% of the patients with BJ and in 49% with MJ.Bilirubin and CA19-9 average levels were significantly higher in MJ compared with BJ(P = 0.000 and P = 0.02),while the CRP level was significantly higher in BJ(P = 0.000).Considering a CA19-9 cut-off level of 32 U/mL,82.3% in the MJ group and 54.9% in the BJ group were positive for CA19-9(P = 0.002).A CA19-9 cut-off of 100 U/mL increases the difference between the two groups:35.3% in BJ and 68.6% in MJ(P = 0.0007).Adjusting the CA19-9 value by dividing it by serum bilirubin level meant that 21.5% in the BJ and 49% in the MJ group remained with a positive CA19-9 value(P = 0.003),while adjusting the CA19-9 value by dividing it by serum CRP value meant that 31.4% in the BJ group and 76.5% in the MJ group still had a positive CA19-9 value(P = 0.000004).Sensitivity,specificity,positive predictive values of CA19-9 > 32 U/mL were 82.3%,45% and 59.1%;when the cutoff was CA19-9 > 100 U/mL they were,respectively,68.6%,64.7% and 66%.When the CA19-9 value was adjusted by dividing it by the bilirubin or CRP values,these became 49%,78.4%,69.4% and 76.5%,68.6%,70.9%,respectively.CONCLUSION:The present study proposes CRP as a new and useful correction factor to improve the diag-nostic value of the CA19-9 tumor marker in patients with cholestatic jaundice.展开更多
Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is ...Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.展开更多
The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the poss...The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 （control group）, 25, 50 and 100 μg/mL （CRP groups） for 24 h. In PDTC （a specific NF-κB inhibitor） group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media （CM） and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α （IκB-α） and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.展开更多
Objective To investigate the role of RNA binding protein—upstream-of-N-Ras(UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect...Objective To investigate the role of RNA binding protein—upstream-of-N-Ras(UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9(RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siR NA was transfected in A172 and T98 G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in highgrade glioma [Grade Ⅱ(n=126), Grade Ⅲ(n=51), Grade Ⅳ(n=128), P<0.001]. UNR high expression levels were associated with poor prognosis(P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples(P<0.01). Knockdown of UNR inhibited glioma cells migration(P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region(UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines(n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression.Epithelial-mesenchymal transition(EMT)-related marker—vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.展开更多
The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reporte...The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.展开更多
Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] di...Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.展开更多
基金the National Natural Science Foundation of China(31730073).
文摘CRISPR/Cas9 technology is a powerful genome manipulation tool in insects.However,little is known about whether mRNA and protein of a target gene are completely cleared in homozygous mutants.This study generated homozygous mutants of the insulin receptor gene 2(NlInR2)in the brown planthopper(Nilaparvata lugens)using CRISPR/Cas9 genome editing.Both frameshift mutants,E5_D17 and E6_I7,differentiated towards long wings,but there were differences in wing morphology,with E5_D17 showing wing deformities.Subsequent investigations revealed the presence of residual expression of NlInR2 mRNA in both mutants,as well as the occurrence of spliceosomes featuring exon skipping splicing in E5_D17.Additionally,the E5_D17 exhibited the detection of N-terminally truncated NlInR2 protein.RNA interference experiments indicated that the knockdown of NlInR2 expression in the E5_D17 mutant line increased the proportion of wing deformities from 11.1 to 65.6%,suggesting that the residual NlInR2 mRNA of the E5_D17 mutant might have retained some genetic functions.Our results imply that systematic characterization of residual protein expression or function in CRISPR/Cas9-generated mutant lines is necessary for phenotypic interpretation.
文摘AIM:To find a possible relationship between inflammation and CA19-9 tumor marker by analyzing data from patients with benign jaundice(BJ) and malignant jaundice(MJ).METHODS:All patients admitted for obstructive jaundice,in the period 2005-2009,were prospectively enrolled in the study,obtaining a total of 102 patients.On admission,all patients underwent complete standard blood test examinations including C-reactive protein(CRP),bilirubin,CA19-9.Patients were considered eligible for the study when they presented obstructive jaundice confirmed by instrumental examinations and increased serum bilirubin levels(total bilirubin > 2.0 mg/dL).The standard cut-off level for CA19-9 was 32 U/mL,whereas for CRP this was 1.5 mg/L.The CA19-9 level was adjusted by dividing it by the value of serum bilirubin or by the CRP value.The patients were divided into 2 groups,MJ and BJ,and after the adjustment a comparison between the 2 groups of patients was performed.Sensitivity,specificity and positive predictive values were calculated before and after the adjustment.RESULTS:Of the 102 patients,51 were affected by BJ and 51 by MJ.Pathologic CA19-9 levels were found in 71.7% of the patients.In the group of 51 BJ patients there were 29(56.9%) males and 22(43.1%) females with a median age of 66 years(range 24-96 years),whereas in the MJ group there were 24(47%) males and 27(53%) females,with a mean age of 70 years(range 30-92 years).Pathologic CA19-9 serum level was found in 82.3% of MJ.CRP levels were pathologic in 66.6% of the patients with BJ and in 49% with MJ.Bilirubin and CA19-9 average levels were significantly higher in MJ compared with BJ(P = 0.000 and P = 0.02),while the CRP level was significantly higher in BJ(P = 0.000).Considering a CA19-9 cut-off level of 32 U/mL,82.3% in the MJ group and 54.9% in the BJ group were positive for CA19-9(P = 0.002).A CA19-9 cut-off of 100 U/mL increases the difference between the two groups:35.3% in BJ and 68.6% in MJ(P = 0.0007).Adjusting the CA19-9 value by dividing it by serum bilirubin level meant that 21.5% in the BJ and 49% in the MJ group remained with a positive CA19-9 value(P = 0.003),while adjusting the CA19-9 value by dividing it by serum CRP value meant that 31.4% in the BJ group and 76.5% in the MJ group still had a positive CA19-9 value(P = 0.000004).Sensitivity,specificity,positive predictive values of CA19-9 > 32 U/mL were 82.3%,45% and 59.1%;when the cutoff was CA19-9 > 100 U/mL they were,respectively,68.6%,64.7% and 66%.When the CA19-9 value was adjusted by dividing it by the bilirubin or CRP values,these became 49%,78.4%,69.4% and 76.5%,68.6%,70.9%,respectively.CONCLUSION:The present study proposes CRP as a new and useful correction factor to improve the diag-nostic value of the CA19-9 tumor marker in patients with cholestatic jaundice.
文摘Age-related NADH oxidase (arNOX = ENOX3) proteins are superoxide-generating cell surface oxidases that increase in activity with age beginning at about 30 y. A soluble and truncated exfoliated form of the activity is present in blood and other body fluids. The activity was purified to apparent homogeneity from human urine and resolved by 2-D gel electrophoresis into a series of 24 to 32 kDa components of low isoelectric point. The purified proteins were resistant both to N-terminal sequencing and trypsin cleavage. Cleavage with pepsin revealed peptides corresponding to the TM9 family of transmembrane proteins. Peptide antisera raised to all five members of the human TM9 family sequentially blocked the arNOX activity of human saliva and sera. The soluble truncated N-terminus of the human homolog TM9SF4 was expressed in bacteria. The recombinant protein was characterized biochemically and exhibited ar-NOX activity. The findings identify five arNOX isoforms each of which correspond to one of the five known TM9 family members. The exfoliated soluble arNOX forms are derived from the 24 to 32 kDa N-termini exposed to the cell’s exterior at the cell surface. Each of the shed forms contain putative functional motifs characteristic of ECTO-NOX (ENOX) proteins despite only minimal sequence identity. Our findings identify arNOX as having functional characteristics of ENOX proteins and the TM9 superfamily of proteins as the genetic origins of the five known arNOX isoforms present in human sera, plasma and other body fluids1.
文摘The relation between the expression and activity of MMP-9 in C-reactive protein （CRP）-induced human THP-1 mononuclear cells and the activation of nuclear factor kappa-B （NF-κB） was studied to investigate the possible role of CRP in plaque destabilization. Human THP-1 cells were incubated in the presence of CRP at 0 （control group）, 25, 50 and 100 μg/mL （CRP groups） for 24 h. In PDTC （a specific NF-κB inhibitor） group, the cells were pre-treated with PDTC at 10 μmol/L and then with 100 μg/mL CRP. The conditioned media （CM） and human THP-1 cells in different groups were harvested. MMP-9 expression in CM and human THP-1 cells was measured by ELISA and Western blotting. MMP-9 activity was assessed by fluorogenic substrates. The expression of NF-κB inhibitor α （IκB-α） and NF-κB p65 was detected by Western blotting and ELISA respectively. The results showed that CRP increased the expression and activity of MMP-9 in a dose-dependent manner in the human THP-1 cells. Western blotting revealed that IiB-α expression was decreased in the cells with the concentrations of CRP and ELISA demonstrated that NF-κB p65 expression in the CRP-induced cells was increased. After pre-treatment of the cells with PDTC at 10 μmol/L, the decrease in IκB-α expression and the increase in NF-κB p65 expression in the CRP-induced cells were inhibited, and the expression and activity of MMP-9 were lowered too. It is concluded that increased expression and activity of MMP-9 in CRP-induced human THP-1 cells may be associated with activation of NF-κB. Down-regulation of the expression and activity of MMP-9 may be a new treatment alternative for plaque stabilization by inhibiting the NF-κB activation.
基金Supported by the CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-001)
文摘Objective To investigate the role of RNA binding protein—upstream-of-N-Ras(UNR) in the development of glioma and its molecular mechanism.Methods First, bioinformatics analysis of CGGA database was performed to detect UNR expression level and prognosis of patients with glioma. Western blot and real-time PCR were used to detect UNR expression level in glioma cell lines and tissues. Next, UNR siRNAs were transfected in glioma cells, and MTS assay and scratch wound-healing assay were used to detect changes in cell proliferation and migration. Then, the candidate UNR target mRNAs were identified by analyzing the sequencing data of UNR iCLIP-seq, RNA sequencing and ribosome profiling databases of human melanoma. RNA immunoprecipitation and biotin pull-down assays were used to identify the UNR target mRNAs in glioma cells. Finally, western blot was used to detect the effect of UNR knockdown on ribosomal protein L9(RPL9) and RPL9 protein expression level in glioma cell lines. RPL9 siR NA was transfected in A172 and T98 G and the expression of vimentin in the cells was detected with western blot.Results Bioinformatics analysis showed that UNR mRNA expression level was significantly higher in highgrade glioma [Grade Ⅱ(n=126), Grade Ⅲ(n=51), Grade Ⅳ(n=128), P<0.001]. UNR high expression levels were associated with poor prognosis(P=0.0177). UNR had high expression level in glioma cell lines and patient samples compared with normal cell lines and normal brain samples(P<0.01). Knockdown of UNR inhibited glioma cells migration(P<0.05), but did not inhibit glioma cells growth in three glioma cell lines. UNR binded the 3' untranslated region(UTR) of PTEN and RPL9 mRNAs. RPL9 protein was significantly highly expressed in most glioma cell lines(n=9) and knockdown of UNR resulted in a downregulation of RPL9 protein expression.Epithelial-mesenchymal transition(EMT)-related marker—vimentin was positively regulated by RPL9.Conclusions UNR could bind to the 3'UTR of PTEN and RPL9 in glioma cell lines, therefore promoting glioma cell migration and regulating the expression of RPL9. Here, we establish a link between UNR and RPL9 protein, which will provide new ideas for the further study of glioma.
基金supported by grants from the Key Project of Guangzhou(No.1561000151)the Yangtze River Scholarship(No.85000-52121100)+1 种基金the National Nature Science Foundation in China(No.81530081,31571395)the 973 project(No.2012CB967000)
文摘The regulation of protein stability is a fundamental issue for biophysical processes,but there has not previously been a convenient and unbiased method of identifying regulators of protein stability.However,as reported in the article entitled "A genome-scale CRISPR-Cas9 screening method for protein stability reveals novel regulators of Cdc25 A," recently published in Cell Discovery,our team developed a protein stability regulators screening assay(Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9(CRISPR-Cas9) library with a dual-fluorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability.Based on our findings,we are confident that this efficient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.
文摘Human μ-opioid receptor （HμOR） with a tag of six consecutive histidines at its carboxyl terminus had been expressed in recombinant baculovirus infected Sf9 insect cells.The maximal binding capacity for the [3H] diprenorphine and [3H]ohmefentanyl （Ohm） were 9.1± 0.7 and 6.52±0.23 nmol/g protein, respectively. The [3H] diprenorphine or [3H] Ohm binding to the receptor expressed in Sf9 cells was strongly inhibited by μ-selective agonists [D-Ala2], N-methylPhe4, glyol5]enkephalin （DAGO）, Ohm, and morphine, but neither by δ nor by K selective agonist. Na+ （100 mM） and GTP （50 μM） could reduce HμOR agonists etorphine and Ohm affinity binding to the overexpressed HμOR. μ-selective agonists DAGO and Ohm effectively stimulated [35S]GTPγS binding (EC50 = 2.7nM and 6.9 nM) and inhibited forskolin- stimulated cAMP accumulation (IC50 = 0.9 nM and 0.3 nM). The agonist-dependent effects could be blocked by opioid antagonist naloxone or by pretreatment of cells with pertussis toxin （PTX）. These results demonstrated that HμOR overexpressed in Sf9 insect cells functionally coupled to endogenous Gi/o proteins.
