The purpose of this paper is to utilize the signaling pathway polymerase chain reaction(PCR)arrays to investigate the activation of two important biological signaling pathways in endothelial cell adhesion and growth m...The purpose of this paper is to utilize the signaling pathway polymerase chain reaction(PCR)arrays to investigate the activation of two important biological signaling pathways in endothelial cell adhesion and growth mediated by adsorbed serum protein on the surface of bare and titanium nitride(TiN)-coated nickel titanium(NiTi)alloys.First,the endothelial cells were cultured on the bare and TiN-coated NiTi alloys and chitosan films as control for 4 h and 24 h,respectively.Then,the total RNA of the cells was collected and the PCR arrays were performed.After that,the differentially expressed genes in the transforming growth factor beta(TGF-b)signaling pathway and the regulation of actin cytoskeleton pathway were screened out;and the further bioinformatics analyses were performed.The results showed that both TGF-b signaling pathway and regulation of actin cytoskeleton pathway were activated in the cells after 4 h and 24 h culturing on the surface of bare and TiN-coated NiTi alloys compared to the chitosan group.The activated TGF-b signaling pathway promoted cell adhesion;the activated regulation of actin cytoskeleton pathway promoted cell adhesion,spreading,growth and motility.In addition,the activation of both pathways was much stronger in the cells cultured for 24 h versus 4 h,which indicated that cell adhesion and growth became more favorable with longer time on the surface of two NiTi alloy materials.展开更多
A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutati...A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.展开更多
Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfl...Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.展开更多
Cancer is a major public health problem throughout the world. It is estimated that one third of the American population will develop the disease at some time during their lifetimes. Among these, melanoma will account ...Cancer is a major public health problem throughout the world. It is estimated that one third of the American population will develop the disease at some time during their lifetimes. Among these, melanoma will account for 7% of the cases. In Brazil, in 2012, it is estimated that over six thousand new melanoma cases occurred. During recent years, the incidence of melanoma has increased, mainly due to a more constant exposure of the skin to sunlight. In this work, our aim is to assess the expression of apoptotis-related genes melanoma tumors in mice treated with Viscum album (VA). This will allow us to better understand the molecular mechanisms underlying tumor cell death activation caused by this compound. Our results clearly demonstrate upregulation of pro apoptotic genes (Trp53bp2, Nol3, Fadd, Tnfsf10, Traf1, Traf2, Cflar, Card10, Nod1, Casp 2, Casp7, Xiap, Dad1, and Dffb). Further bioinformatics-based tool analysis allowed us to assess which specific cell death-related intracellular pathways were activated by VA treatment. Two major effects of VA in melanoma cells could be observed: generation of an immunomudulatory Th-1 like action, recruiting several interleukines, and cell death activation through Caspase7, associated uspstream with Card10 and downstream with CAD. In summary, VA modulates apoptosis related genes in cancer melanoma cells. Although a deeper study should be conducted, VA seems to interfere with important signaling pathways within melanoma cells that control the cellular mechanisms of apoptosis activation. Therapeutic approaches using VA as an antineoplastic and adjuvant medication compounding should be considered.展开更多
Background and Aims:The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain,including its expression pattern and relevance to tumor etiology,tumor stage and prognosi...Background and Aims:The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain,including its expression pattern and relevance to tumor etiology,tumor stage and prognosis,and finally,its impact on apoptosis.Methods:miR-34a expression was assessed in hepatitis C virus (HCV)-induced non-metastatic HCC tissues by RT-Q-PCR.Huh-7 cells were transfected with miR-34a mimics and the impact of miR-34a was examined on 84 pro-apoptotic/anti-apoptotic genes using PCR array;its net effect was tested on cell viability via MTTassay.Results:miR-34a expression was up-regulated in HCC tissues.Moreover,miR-34a induced a large set of pro-apoptotic/anti-apoptotic genes,with a net result of triggering apoptosis and repressing cell viability.Conclusions:HCC-related differential expression of miR-34a could be etiology-based or stage-specific,and low expression of miR-34a may predict poor prognosis.This study's findings also emphasize the role of miR-34a in apoptosis.展开更多
基金National Natural Science Foundation of China(31271012)973 Project(No.2009CB930000)the Natural Science Foundation of Jiangsu Province(BK20150599).
文摘The purpose of this paper is to utilize the signaling pathway polymerase chain reaction(PCR)arrays to investigate the activation of two important biological signaling pathways in endothelial cell adhesion and growth mediated by adsorbed serum protein on the surface of bare and titanium nitride(TiN)-coated nickel titanium(NiTi)alloys.First,the endothelial cells were cultured on the bare and TiN-coated NiTi alloys and chitosan films as control for 4 h and 24 h,respectively.Then,the total RNA of the cells was collected and the PCR arrays were performed.After that,the differentially expressed genes in the transforming growth factor beta(TGF-b)signaling pathway and the regulation of actin cytoskeleton pathway were screened out;and the further bioinformatics analyses were performed.The results showed that both TGF-b signaling pathway and regulation of actin cytoskeleton pathway were activated in the cells after 4 h and 24 h culturing on the surface of bare and TiN-coated NiTi alloys compared to the chitosan group.The activated TGF-b signaling pathway promoted cell adhesion;the activated regulation of actin cytoskeleton pathway promoted cell adhesion,spreading,growth and motility.In addition,the activation of both pathways was much stronger in the cells cultured for 24 h versus 4 h,which indicated that cell adhesion and growth became more favorable with longer time on the surface of two NiTi alloy materials.
文摘A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker, while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction, probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the mutated types of 1896G-A and 1901G-A mutations in HBV were 3.81:1 and 1:3.79 respectively, while, in case of sample B, those were 1:2.89 and 1:3.03 respectively, indicating the presence of point mutations in these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in HBV were 1.26:1 and 1.67:1 respectively. From the above observations, it is evident that the method using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.
基金supported by the National Natural Science Foundation of China [Grant agreement 31502124]the National Science and Technology Major Project of China [Grant agreement 2018ZX10733402]
文摘Objective Antimicrobial resistance(AMR)has become a global concern and is especially severe in China.To effectively and reliably provide AMR data,we developed a new high-throughput real-time PCR assay based on microfluidic dynamic technology,and screened multiple AMR genes in broiler fecal samples.Methods A high-throughput real-time PCR system with an new designed integrated fluidic circuit assay were performed AMR gene detection.A total of 273 broiler fecal samples collected from two geographically separated farms were screened AMR genes.Results The new assay with limits of detection ranging from 40.9 to 8,000 copies/reaction.The sensitivity rate,specificity rate,positive predictive value,negative predictive value and correct indices were 99.30%,98.08%,95.31%,99.79%,and 0.9755,respectively.Utilizing this assay,we demonstrate that AMR genes are widely spread,with positive detection rates ranging from 0 to 97.07%in 273 broiler fecal samples.bla CTX-M,bla TEM,mcr-1,fex A,cfr,optr A,and int I1 showed over 80%prevalence.The dissemination of AMR genes was distinct between the two farms.Conclusions We successfully established a new high-throughput real-time PCR assay applicable to AMR gene surveillance from fecal samples.The widespread existence of AMR genes detected in broiler farms highlights the current and severe problem of AMR.
基金The work was supported by grant number 2010/135938-6 from the Sao Paulo Research Foundation(FAPESP)-Brazil.
文摘Cancer is a major public health problem throughout the world. It is estimated that one third of the American population will develop the disease at some time during their lifetimes. Among these, melanoma will account for 7% of the cases. In Brazil, in 2012, it is estimated that over six thousand new melanoma cases occurred. During recent years, the incidence of melanoma has increased, mainly due to a more constant exposure of the skin to sunlight. In this work, our aim is to assess the expression of apoptotis-related genes melanoma tumors in mice treated with Viscum album (VA). This will allow us to better understand the molecular mechanisms underlying tumor cell death activation caused by this compound. Our results clearly demonstrate upregulation of pro apoptotic genes (Trp53bp2, Nol3, Fadd, Tnfsf10, Traf1, Traf2, Cflar, Card10, Nod1, Casp 2, Casp7, Xiap, Dad1, and Dffb). Further bioinformatics-based tool analysis allowed us to assess which specific cell death-related intracellular pathways were activated by VA treatment. Two major effects of VA in melanoma cells could be observed: generation of an immunomudulatory Th-1 like action, recruiting several interleukines, and cell death activation through Caspase7, associated uspstream with Card10 and downstream with CAD. In summary, VA modulates apoptosis related genes in cancer melanoma cells. Although a deeper study should be conducted, VA seems to interfere with important signaling pathways within melanoma cells that control the cellular mechanisms of apoptosis activation. Therapeutic approaches using VA as an antineoplastic and adjuvant medication compounding should be considered.
文摘Background and Aims:The role of miR-34a in hepatocellular carcinoma (HCC) is controversial and several unresolved issues remain,including its expression pattern and relevance to tumor etiology,tumor stage and prognosis,and finally,its impact on apoptosis.Methods:miR-34a expression was assessed in hepatitis C virus (HCV)-induced non-metastatic HCC tissues by RT-Q-PCR.Huh-7 cells were transfected with miR-34a mimics and the impact of miR-34a was examined on 84 pro-apoptotic/anti-apoptotic genes using PCR array;its net effect was tested on cell viability via MTTassay.Results:miR-34a expression was up-regulated in HCC tissues.Moreover,miR-34a induced a large set of pro-apoptotic/anti-apoptotic genes,with a net result of triggering apoptosis and repressing cell viability.Conclusions:HCC-related differential expression of miR-34a could be etiology-based or stage-specific,and low expression of miR-34a may predict poor prognosis.This study's findings also emphasize the role of miR-34a in apoptosis.
