Objective: To evaluate the value of ligase chainreaction(LCR) in the diagnosis of diplococcusgonorrhoeae in urine. Methods: LCR detection of the urine for Neisseriagonorrhoeae and bacteria culture of discharge was per...Objective: To evaluate the value of ligase chainreaction(LCR) in the diagnosis of diplococcusgonorrhoeae in urine. Methods: LCR detection of the urine for Neisseriagonorrhoeae and bacteria culture of discharge was per-formed simultaneously to 276 patients with urethritisor cervicitis seeking treatment in sex transmitted dis-eases (STDs) outpatient clinic. For specimens withdiscordant results, polymerase chain reaction wasconducted. The purpose was to detect the respectivesensitivity and specificity of bacteria culture and LCR. Results: 24 of 276(8.7%) patients had positive LCRresults and 21 of 276(7.6%) were positive for culture.5 specimens had discordant results from LCR andbacteria culture. The sensitivity and specificity of LCRin the diagnosis of gonorrhoeae were 92.3% and100% respectively. Conclusion: This study showed that LCR had ahigher sensitivity and specificity for the diagnosis ofgonorrhoeae from urine.展开更多
Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthes...Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.展开更多
Oral lichen planus （OLP） is a chronic inflammatory disorder and premalignantlesion, of which the mechanisms are still obscure. In the present study, the expression levels of miR-96/182/183 cluster, miR-203, miR-375,...Oral lichen planus （OLP） is a chronic inflammatory disorder and premalignantlesion, of which the mechanisms are still obscure. In the present study, the expression levels of miR-96/182/183 cluster, miR-203, miR-375, and miR-769-5p in both tissues and exfoliative cells of OLP patients as well as healthy volunteers were detected, differentially expressed miRNAs were identified and their correlation with OLP was evaluated by a biplot method. Experimental results show that miR-203 is significantly up-regulated in patient lesion tissues in comparison to volunteer mucosa tissues. Moreover, the contra- dictory insignificant expression changes of miR-203 as well as miR-96/182/183 cluster in comparisons of exfoliative cell samples suggest that different cell compositions in OLP lesion have distinct miRNA regulation, which accords with the histological heterogeneity of OLP. Finally, biplot analyses indicate the expression of miR-203 and miR-96/182/183 cluster are positively correlated in patient lesions. These results provide miR-203 as a molecular indicator of heterogeneity of OLP, and also a potential diagnostic biomarker or therapeutic target that deserves further studies.展开更多
RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcript...RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle and formed a double-stranded DNA in which one terminus was an RNA/DNA hybrid.Because there is a higher sensitivity of the DNA polymerase to the mismatch at the 3?-end in the RNA/DNA hybrid duplex than in the DNA/DNA duplex,the RNA-primed PCR reveals much better specificity in the allele-specific PCR to detect single-nucleotide mutation.展开更多
Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymer...Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase(Pol B), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7 d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of Pol B and Pol D from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPf A1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both Pol B and Pol D were efficient in strand displacement. HPf A1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPf A1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.展开更多
One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosenso...One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosensor was based on ECL photo-quenching effect of ferrocene （Fc） to tris（2,2-bipyridyl）ruthenium（II） （Ru（bpy）2＋）. It was built up by modification of Au nanoparticles （AuNPs） and Ru（bpy）32＋ on one Au electrode firstly, and then self-assembly of one special double-stranded DNA （dsDNA） onto the electrode. This dsDNA was prepared by hybridization of one Fc labeled molecular beacon single-stranded DNA（ssDNA） and one anti-thrombin aptamer ssDNA. Without the target protein, this Fc-dsDNA/Ru（bpy）2＋- AuNPs/Au elec- trode trigged strong ECL signal, so we called it ECL ＂signal on＂ state. When thrombin was present in the sensing solution, the protein reacted with its aptamer from the Fc-dsDNA/Ru（bpy）3^2＋-AuNPs/Au electrode. Then the left molecular beacon ssDNA on the electrode recovered to its normal stem-loop structure and consequently its Fc labeler was close enough to the electrode surface to quench the ECL signal from Ru（bpy）3^2＋. It was in ECL ＂signal off＂ state. We measured the decrease in ECL intensity to sense the target protein. This was one endeavour to sense protein by using un-labeling target or probe strategy, which gave higher sensitivity and selectivity due to the better combination efficiency of protein and the un-labeled aptamer. 6.25 fmo/L thrombin was detected out,展开更多
The ubiquitin （Ub）-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance （or post-replication repair） via catalyzing Lys63-linked polyubiquitin chains formation together with ...The ubiquitin （Ub）-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance （or post-replication repair） via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especial y in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction （PCR） indicated that OsUbc13 transcripts could be de-tected in al tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma;the expression of OsUbc13 was induced by low temperature, methylmethane sulfate （MMS）, and H2O2, but repressed by mannitol, abscisic acid （ABA）, and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC （correlated with apoptosis）, OsMADS1 （important for development of floral organs）, OsB22EL8 （related to reactive oxygen species （ROS） scavenging and DNA protection）, and OsCROC-1 （required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance）. The molecular characterization provides a foundation for the functional study of OsUbc13.展开更多
Revolutionary in scope and application, the CRISPR Cas9 endonuclease system can be guided by 20-nt single guide RNA （sgRNA） to any complementary loci on the double- stranded DNA. Once the target site is located, Cas...Revolutionary in scope and application, the CRISPR Cas9 endonuclease system can be guided by 20-nt single guide RNA （sgRNA） to any complementary loci on the double- stranded DNA. Once the target site is located, Cas9 can then cleave the DNA and introduce mutations. Despite the power of this system, sgRNA is highly susceptible to off-target homologous attachment and can consequently cause Cas9 to cleave DNA at off- target sites. In order to better understand this flaw in the system, the human genome and Streptococcus pyogenes Cas9 （SpCas9） were used in a mathematical and computational study to analyze the probabilities of potential sgRNA off-target homologies. It has been concluded that off-target sites are nearly unavoidable for large-size genomes, such as the human genome. Backed by mathematical analysis, a viable solution is the double-nicking method which has the promise for genome editing specificity. Also applied in this study was a computational algorithm for off-target homology search that was implemented in Java to confirm the mathematical analysis.展开更多
文摘Objective: To evaluate the value of ligase chainreaction(LCR) in the diagnosis of diplococcusgonorrhoeae in urine. Methods: LCR detection of the urine for Neisseriagonorrhoeae and bacteria culture of discharge was per-formed simultaneously to 276 patients with urethritisor cervicitis seeking treatment in sex transmitted dis-eases (STDs) outpatient clinic. For specimens withdiscordant results, polymerase chain reaction wasconducted. The purpose was to detect the respectivesensitivity and specificity of bacteria culture and LCR. Results: 24 of 276(8.7%) patients had positive LCRresults and 21 of 276(7.6%) were positive for culture.5 specimens had discordant results from LCR andbacteria culture. The sensitivity and specificity of LCRin the diagnosis of gonorrhoeae were 92.3% and100% respectively. Conclusion: This study showed that LCR had ahigher sensitivity and specificity for the diagnosis ofgonorrhoeae from urine.
文摘Plant RNA N-glycosidase specifically hydrolyzes the N-C glycosidic bond of a conserved adenosine in the sarcin/ricin domain of the largest RNA in ribosome, releasing an adenine base and thus inhibiting protein synthesis. This substrate specificity was challenged later by discovery that various RNA derivatives and DNAs, especially the double-stranded supercoiled DNA could be used as substrate by RNA N-glycosidase. Thus, it was argued whether the DNA-cleaving activity was an intrinsic feature of RNA N-glycosidase or it was contaminated by DNase. In this article, several lines of evidence are presented to show that RNA N-glycosidase can really release the adenine base from the double-stranded supercoi/ed DNA. It was proposed that the cleavage mechanism of supercoiled DNA was the phosphodiester bonds in enzymatically deadenylated regions of the supercoiled DNA would become fragile and liable to produce nicked or linear form owing to the existence of tension in the supercoiled DNA molecule, not direct result of enzymatic action on the phosphodiester bond.
基金National Natural Science Foundation of China(Grant No.91029711)
文摘Oral lichen planus （OLP） is a chronic inflammatory disorder and premalignantlesion, of which the mechanisms are still obscure. In the present study, the expression levels of miR-96/182/183 cluster, miR-203, miR-375, and miR-769-5p in both tissues and exfoliative cells of OLP patients as well as healthy volunteers were detected, differentially expressed miRNAs were identified and their correlation with OLP was evaluated by a biplot method. Experimental results show that miR-203 is significantly up-regulated in patient lesion tissues in comparison to volunteer mucosa tissues. Moreover, the contra- dictory insignificant expression changes of miR-203 as well as miR-96/182/183 cluster in comparisons of exfoliative cell samples suggest that different cell compositions in OLP lesion have distinct miRNA regulation, which accords with the histological heterogeneity of OLP. Finally, biplot analyses indicate the expression of miR-203 and miR-96/182/183 cluster are positively correlated in patient lesions. These results provide miR-203 as a molecular indicator of heterogeneity of OLP, and also a potential diagnostic biomarker or therapeutic target that deserves further studies.
基金supported by the Chinese Academy of Sciences(Hundreds of Talents Program)the National Natural Science Foundation of China(21172215 and 21102139)the Innovation Program of the Chinese Academy of Sciences(KSCX2-EW-J-22)
文摘RNA/DNA primer pairs and two polymerases were used to efficiently amplify DNA sequences using the conventional polymerase chain reaction(PCR).The reaction required the use of both DNA polymerase and reverse transcriptase during each thermal cycle and formed a double-stranded DNA in which one terminus was an RNA/DNA hybrid.Because there is a higher sensitivity of the DNA polymerase to the mismatch at the 3?-end in the RNA/DNA hybrid duplex than in the DNA/DNA duplex,the RNA-primed PCR reveals much better specificity in the allele-specific PCR to detect single-nucleotide mutation.
基金supported by the National Natural Science Foundation of China (31130003, 30921065)
文摘Euryarchaeota and Crenarchaeota, the two main lineages of the domain Archaea, encode different chromatin proteins and differ in the use of replicative DNA polymerases. Crenarchaea possess a single family B DNA polymerase(Pol B), which is capable of strand displacement modulated by the chromatin proteins Cren7 and Sul7 d. Euryarchaea have two distinct replicative DNA polymerases, PolB and PolD, a family D DNA polymerase. Here we characterized the strand displacement activities of Pol B and Pol D from the hyperthermophilic euryarchaeon Pyrococcus furiosus and investigated the influence of HPf A1, a homolog of eukaryotic histones from P. furiosus, on these activities. We showed that both Pol B and Pol D were efficient in strand displacement. HPf A1 inhibited DNA strand displacement by both DNA polymerases but exhibited little effect on the displacement of a RNA strand annealed to single-stranded template DNA. This is consistent with the finding that HPf A1 bound more tightly to double-stranded DNA than to a RNA:DNA hybrid. Our results suggest that, although crenarchaea and euryarchaea differ in chromosomal packaging, they share similar mechanisms in modulating strand displacement by DNA polymerases during lagging strand DNA synthesis.
文摘One solid-state electrochemiluminescence （ECL） protein biosensor based on the competing reaction and substitute reaction between protein-to-DNA aptamer and DNA-to-DNA aptamer was proposed. Additionally, the biosensor was based on ECL photo-quenching effect of ferrocene （Fc） to tris（2,2-bipyridyl）ruthenium（II） （Ru（bpy）2＋）. It was built up by modification of Au nanoparticles （AuNPs） and Ru（bpy）32＋ on one Au electrode firstly, and then self-assembly of one special double-stranded DNA （dsDNA） onto the electrode. This dsDNA was prepared by hybridization of one Fc labeled molecular beacon single-stranded DNA（ssDNA） and one anti-thrombin aptamer ssDNA. Without the target protein, this Fc-dsDNA/Ru（bpy）2＋- AuNPs/Au elec- trode trigged strong ECL signal, so we called it ECL ＂signal on＂ state. When thrombin was present in the sensing solution, the protein reacted with its aptamer from the Fc-dsDNA/Ru（bpy）3^2＋-AuNPs/Au electrode. Then the left molecular beacon ssDNA on the electrode recovered to its normal stem-loop structure and consequently its Fc labeler was close enough to the electrode surface to quench the ECL signal from Ru（bpy）3^2＋. It was in ECL ＂signal off＂ state. We measured the decrease in ECL intensity to sense the target protein. This was one endeavour to sense protein by using un-labeling target or probe strategy, which gave higher sensitivity and selectivity due to the better combination efficiency of protein and the un-labeled aptamer. 6.25 fmo/L thrombin was detected out,
基金Project supported by the National High-Tech R&D Program(863)of China(No.2006AA10Z159)the National Natural Science Foundation of China(No.30871502)
文摘The ubiquitin （Ub）-conjugating enzyme, Ubc13, has been known to be involved in error-free DNA damage tolerance （or post-replication repair） via catalyzing Lys63-linked polyubiquitin chains formation together with a Ubc variant. However, its functions remain largely unknown in plant species, especial y in monocotyledons. In this study, we cloned a Ub-conjugating enzyme, OsUbc13, that shares the conserved domain of Ubc with AtUBC13B in Oryza sativa L., which encodes a protein of 153 amino acids; the deduced sequence shares high similarities with other homologs. Real-time quantitative polymerase chain reaction （PCR） indicated that OsUbc13 transcripts could be de-tected in al tissues examined, and the expression level was higher in palea, pistil, stamen, and leaf, and lower in root, stem, and lemma;the expression of OsUbc13 was induced by low temperature, methylmethane sulfate （MMS）, and H2O2, but repressed by mannitol, abscisic acid （ABA）, and NaCl. OsUbc13 was probably localized in the plasma and nuclear membranes. About 20 proteins, which are responsible for the positive yeast two-hybrid interaction of OsUbc13, were identified. These include the confirmed OsVDAC （correlated with apoptosis）, OsMADS1 （important for development of floral organs）, OsB22EL8 （related to reactive oxygen species （ROS） scavenging and DNA protection）, and OsCROC-1 （required for formation of Lys63 polyubiquitylation and error-free DNA damage tolerance）. The molecular characterization provides a foundation for the functional study of OsUbc13.
文摘Revolutionary in scope and application, the CRISPR Cas9 endonuclease system can be guided by 20-nt single guide RNA （sgRNA） to any complementary loci on the double- stranded DNA. Once the target site is located, Cas9 can then cleave the DNA and introduce mutations. Despite the power of this system, sgRNA is highly susceptible to off-target homologous attachment and can consequently cause Cas9 to cleave DNA at off- target sites. In order to better understand this flaw in the system, the human genome and Streptococcus pyogenes Cas9 （SpCas9） were used in a mathematical and computational study to analyze the probabilities of potential sgRNA off-target homologies. It has been concluded that off-target sites are nearly unavoidable for large-size genomes, such as the human genome. Backed by mathematical analysis, a viable solution is the double-nicking method which has the promise for genome editing specificity. Also applied in this study was a computational algorithm for off-target homology search that was implemented in Java to confirm the mathematical analysis.
