Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA ex...Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.展开更多
The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application
Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatoc...Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing ccc DNA reservoir. Therefore, the study of the molecular mechanism of ccc DNA formation is becoming a major focus of HBV research. This review summarizes the current advances in ccc DNA molecular biology and the latest studies on the elimination or inactivation of ccc DNA, including three major areas:(1) epigenetic regulation of ccc DNA by HBV X protein,(2) immune-mediated degradation,and(3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.展开更多
Hepatitis B virus(HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed cir...Hepatitis B virus(HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA(ccc DNA), which persists in hepatocyte nuclei. As HBV ccc DNA is a viral transcription template, novel therapeutic approaches to directly target HBV ccc DNA are necessary to completely eradicate persistent HBV infections. HBV ccc DNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequencespecific nuclease with high flexibility and may be the most feasible approach to target HBV ccc DNA. Further research to develop easier, safer, and more effective protocols should be pursued.展开更多
AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.METHODS： Seventy-one patients received lam...AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.METHODS： Seventy-one patients received lamivudine （n = 35）, or sequential therapy with lamivudine- interferon alpha 2b （IFN-α 2b, n = 24） for 48 wk, or IFN-α 2b （n = 12） for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.RESULTS： Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively （P 〈 0.05）. Seventeen out of the 71 patieots developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients （3.0 log vs 1.6 log, P = 0.0407）. Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound （4.6 log vs 5.4 log, P = 0.0472）. HBV genotype C accounted for 85.9% （n = 61）, and genotype B for 14.1% （n = 10）, respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B （2.1 log vs 1.9 log）.CONCLUSION： Forty-eight week sequential lamivudine- INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.展开更多
250 million people worldwide continue to be chronically infected with the virus.While patients may be treated with nucleoside/nucleotide analogues,this only suppresses HBV titre to sub-detection levels without elimina...250 million people worldwide continue to be chronically infected with the virus.While patients may be treated with nucleoside/nucleotide analogues,this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA(cccDNA)genome.As a result,HBV infection cannot be cured,and the virus reactivates when conditions are favorable.Interferons(IFNs)are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells.They have been shown to induce cccDNA clearance,but their use in the treatment of HBV infection is limited as HBVtargeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling.Thus,to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV,instead of direct IFN administration,novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed.This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions.These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications.In addition,the mechanisms that HBV employs to disrupt IFN signalling will be discussed.Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections.Together,these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.展开更多
Chronic infection with hepatitis B virus(HBV)remains a major global health problem,especially in developing countries.It may lead to prolonged liver damage,fibrosis,cirrhosis,and hepatocellular carcinoma.Persistent ch...Chronic infection with hepatitis B virus(HBV)remains a major global health problem,especially in developing countries.It may lead to prolonged liver damage,fibrosis,cirrhosis,and hepatocellular carcinoma.Persistent chronic HBV infection is related to host immune response and the stability of the covalently closed circular DNA(cccDNA)in human hepatocytes.In addition to being essential for viral transcription and replication,cccDNA is also suspected to play a role in persistent HBV infections or hepatitis relapses since cccDNA is very stable in non-dividing human hepatocytes.Understanding the pathogenicity and oncogenicity of HBV components would be essential in the development of new diagnostic tools and treatment strategies.This review summarizes the role and molecular mechanisms of HBV cccDNA in hepatocyte transformation and hepatocarcinogenesis and current efforts to its detection and targeting.展开更多
Binding of cordycepin to the double helical DNA with a high affinity was investigated by CD spectra in this paper. The results proved that uncoiling, unbinding and denaturation of DNA proceeded continuously upon the i...Binding of cordycepin to the double helical DNA with a high affinity was investigated by CD spectra in this paper. The results proved that uncoiling, unbinding and denaturation of DNA proceeded continuously upon the increase of the concentration of cordycepin.展开更多
BACKGROUND Minimally invasive or noninvasive,sensitive and accurate detection of colorectal cancer(CRC)is urgently needed in clinical practice.AIM To identify a noninvasive,sensitive and accurate circular free DNA mar...BACKGROUND Minimally invasive or noninvasive,sensitive and accurate detection of colorectal cancer(CRC)is urgently needed in clinical practice.AIM To identify a noninvasive,sensitive and accurate circular free DNA marker detected by digital polymerase chain reaction(dPCR)for the early diagnosis of clinical CRC.METHODS A total of 195 healthy control(HC)individuals and 101 CRC patients(38 in the early CRC group and 63 in the advanced CRC group)were enrolled to establish the diagnostic model.In addition,100 HC individuals and 62 patients with CRC(30 early CRC and 32 advanced CRC groups)were included separately to validate the model.CAMK1D was dPCR.Binary logistic regression analysis was used to establish a diagnostic model including CAMK1D and CEA.RESULTS To differentiate between the 195 HCs and 101 CRC patients(38 early CRC and 63 advanced CRC patients),the common biomarkers CEA and CAMK1D were used alone or in combination to evaluate their diagnostic value.The area under the curves(AUCs)of CEA and CAMK1D were 0.773(0.711,0.834)and 0.935(0.907,0.964),respectively.When CEA and CAMK1D were analyzed together,the AUC was 0.964(0.945,0.982).In differentiating between the HC and early CRC groups,the AUC was 0.978(0.960,0.995),and the sensitivity and specificity were 88.90%and 90.80%,respectively.In differentiating between the HC and advanced CRC groups,the AUC was 0.956(0.930,0.981),and the sensitivity and specificity were 81.30%and 95.90%,respectively.After building the diagnostic model containing CEA and CAMK1D,the AUC of the CEA and CAMK1D joint model was 0.906(0.858,0.954)for the validation group.In differentiating between the HC and early CRC groups,the AUC was 0.909(0.844,0.973),and the sensitivity and specificity were 93.00%and 83.30%,respectively.In differentiating between the HC and advanced CRC groups,the AUC was 0.904(0.849,0.959),and the sensitivity and specificity were 93.00%and 75.00%,respectively.CONCLUSION We built a diagnostic model including CEA and CAMK1D for differentiating between HC individuals and CRC patients.Compared with the common biomarker CEA alone,the diagnostic model exhibited significant improvement.展开更多
Fur seal feces-associated circular DNA virus(FSfa CV)is an unclassified circular replication-associated protein(Rep)-encoding single-stranded(CRESS)DNA virus that has been detected in mammals(fur seals and pigs).The b...Fur seal feces-associated circular DNA virus(FSfa CV)is an unclassified circular replication-associated protein(Rep)-encoding single-stranded(CRESS)DNA virus that has been detected in mammals(fur seals and pigs).The biology and epidemiology of the virus remain largely unknown.To investigate the virus diversity among pigs in Anhui Province,China,we pooled 600 nasal samples in 2017 and detected viruses using viral metagenomic methods.From the assembled contigs,12 showed notably high nucleotide acid sequence similarities to the genome sequences of FSfa CVs.Based on these sequences,a full-length genome sequence of the virus was then obtained using overlapping PCR and sequencing,and the virus was designated as FSfa CV-CHN(Gen Bank No.MK462122).This virus shared 91.3%and 90.9%genome-wide nucleotide sequence similarities with the New Zealand fur seal strain FSfa CV-as50 and the Japanese pig strain FSfa CVJPN1,respectively.It also clustered with the two previously identified FSfa CVs in a unique branch in the phylogenetic tree based on the open reading frame 2(ORF2),Rep-coding gene,and the genome of the reference CRESS DNA viruses.Further epidemiological investigation using samples collected in 2018 showed that the overall positive rate for the virus was 56.4%(111/197)in Anhui Province.This is the first report of FSfa CVs identified in pigs in China,and further epidemiological studies are warranted to evaluate the influence of the virus on pigs.展开更多
BACKGROUND Colorectal cancer(CRC) is the third most common cancer worldwide, and it is the second leading cause of death from cancer in the world, accounting for approximately 9% of all cancer deaths. Early detection ...BACKGROUND Colorectal cancer(CRC) is the third most common cancer worldwide, and it is the second leading cause of death from cancer in the world, accounting for approximately 9% of all cancer deaths. Early detection of CRC is urgently needed in clinical practice.AIM To build a multi-parameter diagnostic model for early detection of CRC.METHODS Total 59 colorectal polyps(CRP) groups, and 101 CRC patients(38 early-stage CRC and 63 advanced CRC) for model establishment. In addition, 30 CRP groups,and 62 CRC patients(30 early-stage CRC and 32 advanced CRC) were separately included to validate the model. 51 commonly used clinical detection indicators and the 4 extrachromosomal circular DNA markers NDUFB7, CAMK1D, PIK3CD and PSEN2 that we screened earlier. Four multi-parameter joint analysis methods:binary logistic regression analysis, discriminant analysis, classification tree and neural network to establish a multi-parameter joint diagnosis model.RESULTS Neural network included carcinoembryonic antigen(CEA), ischemia-modified albumin(IMA),sialic acid(SA), PIK3CD and lipoprotein a(LPa) was chosen as the optimal multi-parameter combined auxiliary diagnosis model to distinguish CRP and CRC group, when it differentiated 59CRP and 101 CRC, its overall accuracy was 90.8%, its area under the curve(AUC) was 0.959(0.934,0.985), and the sensitivity and specificity were 91.5% and 82.2%, respectively. After validation,when distinguishing based on 30 CRP and 62 CRC patients, the AUC was 0.965(0.930-1.000), and its sensitivity and specificity were 66.1% and 70.0%. When distinguishing based on 30 CRP and 32early-stage CRC patients, the AUC was 0.960(0.916-1.000), with a sensitivity and specificity of 87.5% and 90.0%, distinguishing based on 30 CRP and 30 advanced CRC patients, the AUC was 0.970(0.936-1.000), with a sensitivity and specificity of 96.7% and 86.7%.CONCLUSION We built a multi-parameter neural network diagnostic model included CEA, IMA, SA, PIK3CD and LPa for early detection of CRC, compared to the conventional CEA, it showed significant improvement.展开更多
Knots are discovered in biophysical systems,such as DNA and proteins.Knotted portions in knotted DNA are significantly bent and their corresponding bending angles are comparable with or larger than the sharp bending a...Knots are discovered in biophysical systems,such as DNA and proteins.Knotted portions in knotted DNA are significantly bent and their corresponding bending angles are comparable with or larger than the sharp bending angle resulting in flexible defects.The role of flexible defects in the interplay of supercoiling and knotting of circular DNA were predicted by a Monte Carlo simulation.In knotted DNA with a particular knot type,a flexible defect noticeably enhances the supercoiling of the knotted DNA and the decreasing excitation energy makes the knotted portion more compact.A reduction in twist rigidity and unwinding of flexible defects are incorporated into the numerical simulations,so that interplay of supercoiling and knotting of circular DNA is studied under torsional conditions.Increasing unwinding not only results in a wider linking number distribution,but also leads to a drift of the distribution to lower values.A flexible defect has obvious effects on knotting probability.The summation of equilibrium distribution probability for nontrivial knotted DNA with different contour length does not change with excitation energy monotonically and has a maximum at an intermediate value of excitation energy around 5 kBT.In the phase space of knot length and gyration radius of knotted DNA,knot length does not anticorrelate with its gyration radius,which is attributed to the flexible defect in the knotted portion,which leads to the release of bending energy and inhibited the competition between entropy and bending energy.展开更多
To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of ...To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.展开更多
AIM: To investigate the reduction in hepatitis B virus(HBV) covalently closed-circular DNA(ccc DNA) with entecavir(ETV) or lamivudine(LAM). METHODS: This analysis included patients who had participated in the randomiz...AIM: To investigate the reduction in hepatitis B virus(HBV) covalently closed-circular DNA(ccc DNA) with entecavir(ETV) or lamivudine(LAM). METHODS: This analysis included patients who had participated in the randomized Phase Ⅲ study ETV-022 comparing ETV vs LAM in nucleos(t)ide-naive, HBe Agpositive patients. Patients received ETV(0.5 mg daily) or LAM(100 mg daily) for a minimum of 52 wk. Patients were eligible to participate in this sub-study if they had paired biopsies at baseline and week 48 with evaluable measurements for hepatic HBV ccc DNA and total hepatic HBV DNA. The main objective was to compare changes in hepatic HBV ccc DNA and total hepatic HBV DNA at week 48 of ETV or LAM treatment, which was a secondary endpoint of study ETV-022. Additional post hoc analyses included linear regression analyses to assess associations of baseline levels and on-treatment changes of ccc DNA with other baseline factors [sex,age, serum HBV DNA, alanine aminotransferase(ALT), Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBV genotype], or ontreatment factors(changes from baseline at week 48 in serum HBV DNA, ALT, Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBe Ag loss at week 48).RESULTS: Overall, 305 patients(ETV = 159; LAM = 146) of ETV-022 had paired baseline and week 48 liver biopsies with evaluable measurements for hepatic HBV ccc DNA and total hepatic HBV DNA, and were included in this analysis. Baseline demographics and disease characteristics were comparable between the two arms. After 48 wk, ETV resulted in significantly greater reductions in hepatic HBV ccc DNA [-0.9 log10 copies/human genome equivalent(HGEq) vs-0.7 log10 copies/HGEq; P = 0.0033] and total hepatic DNA levels(-2.1 log10 copies/HGEq vs-1.6 log10 copies/HGEq; P < 0.0001) than LAM. Virologic, biochemical, and histologic response rates at week 48 were also greater with ETV than with LAM. Baseline HBV ccc DNA levels were positively associated with baseline levels of serum HBV DNA and total hepatic HBV DNA, and negatively associated with HBV genotype F. On-treatment changes in HBV ccc DNA levels were negatively associated with baseline levels of serum HBV DNA and baseline ALT, and were positively associated with on-treatment changes in the levels of serum HBV DNA, total hepatic HBV DNA levels, and ALT, change in Knodell necroinflammatory score, and HBe Ag loss.CONCLUSION: Forty-eight weeks of ETV resulted in greater reductions in ccc DNA and total hepatic HBV DNA than LAM, but long-term therapy may be needed for ccc DNA elimination.展开更多
目的运用meta分析方法系统评价补肾方剂治疗男性不育症患者精子DNA损伤(SDF)的有效性和安全性。方法从中国知网、维普、万方、PubMed、Cochrane Library、Embase、Web of Science数据库中检索补肾方剂改善SDF的随机对照试验(RCT)研究,...目的运用meta分析方法系统评价补肾方剂治疗男性不育症患者精子DNA损伤(SDF)的有效性和安全性。方法从中国知网、维普、万方、PubMed、Cochrane Library、Embase、Web of Science数据库中检索补肾方剂改善SDF的随机对照试验(RCT)研究,对文献进行方法学质量评价,运用RevMan5.3.5软件进行meta分析,并对结局指标进行GRADE质量分级。结果共纳入17项RCT研究,涉及2164例男性患者。与西医常规治疗相比,补肾方剂能够显著改善男性不育症患者DNA碎片指数(DFI)(MD=-6.50,95%CI:-7.88~-5.11,P<0.01),提高配偶妊娠率(RR=2.11,95%CI:1.12~4.00,P=0.87)、精子总活率(MD=5.56,95%CI:4.39~6.74,P<0.01)、前向运动精子百分率(MD=6.82,95%CI:5.62~8.03,P<0.01)、精子浓度(MD=6.51,95%CI:3.81~9.21,P<0.01)和正常形态精子百分率(MD=1.26,95%CI:0.45~2.06,P<0.01),且补肾方剂不会增加不良反应的发生。结论低到中等质量证据表明,与西医常规治疗相比,补肾方剂在改善男性不育症患者DFI、精液参数、配偶妊娠率等方面具有一定优势,且安全性较好。展开更多
The different topological states of circular double-stranded DNA can be defined by their linking number. The equilibrium distribution of linking number can be obtained by circularizing a linear DNA into a circle by li...The different topological states of circular double-stranded DNA can be defined by their linking number. The equilibrium distribution of linking number can be obtained by circularizing a linear DNA into a circle by ligase. Based on the recent experimental results that the DNA bending rigidity and twist rigidity strongly depend on temperature, the reduced bending rigidity can be approximated by g = （3.19 x10-19 - T. 4.14s10-22） erg. cm over the temperature interval （5 ~ 53） ~ C, and the temperature dependence of twist rigidity can be fitted by C （ T） = （4588.89 exp（-T/117.04）- 251.33） nm. The temperature dependence of the linking number distribution of circular DNAs can be predicted by using Monte Carlo simulation. The variance of linking number distribution on temperature is in accordance with the previous experimental results. Compared with the temperature dependence of bending rigidity, the temperature dependence of twist rigidity causes a noticeable fluctuation in linking number distribution and mainly contribute towards the variance change of linking number distribution of circular DNA. The variance of the writhe number and twist number in the equation （（ALk）21 = （（ATw）2） -b （（Wr）2） depends on the length of circular DNA. When the length of circular DNA is less than 230 nm, the variance of twist number （（ATw）2） is dominant over the variance of writhe number （（（wr）2））whereas for the condition that the length of the circular DNA is larger than 370 nm.展开更多
Circular dichroism (CD) is a reliable,sensitiveand convenient methods for analysing the effectof drugs on DNA,which intercalated into theDNA molecule or formed DNA complex and couldchange the conformation of DNA and a...Circular dichroism (CD) is a reliable,sensitiveand convenient methods for analysing the effectof drugs on DNA,which intercalated into theDNA molecule or formed DNA complex and couldchange the conformation of DNA and alter theCD spectra.Mutagenicity of 19 drugs展开更多
基金Project supported by the Exchange Scholarship Programs of the Landesregierung Schleswig-Holstein,Germany,for Jian-er WO
文摘Objective: This study aimed to investigate DNA sequences that are substantially homologous to the corresponding RNA sequence sections of the hepatitis C virus (HCV). These DNA sequences are present in the whole DNA extracted from peripheral blood mononuclear cells (PBMCs) of HCV-negative subjects. We presumed that these experimentally proven 5'-noncoding region (5'-NCR) homologous DNA sequences could be contained in the extrachromosomal circular DNA (eccDNA) fraction as part of the whole cellular DNA. Methods: Home-made polymerase chain reaction (PCR) with whole cellular and isolated eccDNA, nucleotide basic local alignment search tool (BLASTn) alignments, and tests for patterns of methylation in selected sequence sections were performed. Results: The PCR tests revealed DNA sequences of up to 320 bp that broadly matched the corresponding sequence sections of known HCV genotypes. In contrast, BLASTn alignment searches of published HCV 5'-NCR sequences with human genome databases revealed only sequence segments of up to 36 bp of the 5'-NCR. The composition of these sequences shows missing base pairs, base pair mismatches as well as complete homology with HCV reference sequences. These short sequence sections are present in numerous copies on both the same and different chromosomes. The selected sequence region within the DNA sequences of the 5'-NCR revealed a broad diversity of individual patterns of methylation. Conclusions: The experimental results confirm our assumption that parts of the HCV 5'-NCR genomic RNA sequences are present at the DNA level in the eccDNA fraction of PBMCs. The tests for methylation patterns therein revealed individual methylomes which could represent an epigenetic feature. The respective sequence section might be subject to genetic regulation.
文摘The availability of the plastid genome sequences is one of the bases for comparative,functional,and structural genomic studies of plastid-containing living organisms,in addition to the application
基金supported by the Key Project of Hubei Province Natural Science Foundation(2014CFA075)the National Natural Science Foundation of China(31400153)the Applied Basic Research Program(2015060101010033),Wuhan,China
文摘Chronic hepatitis B infection is caused by hepatitis B virus(HBV) and a total cure is yet to be achieved. The viral covalently closed circular DNA(ccc DNA) is the key to establish a persistent infection within hepatocytes. Current antiviral strategies have no effect on the pre-existing ccc DNA reservoir. Therefore, the study of the molecular mechanism of ccc DNA formation is becoming a major focus of HBV research. This review summarizes the current advances in ccc DNA molecular biology and the latest studies on the elimination or inactivation of ccc DNA, including three major areas:(1) epigenetic regulation of ccc DNA by HBV X protein,(2) immune-mediated degradation,and(3) genome-editing nucleases. All these aspects provide clues on how to finally attain a cure for chronic hepatitis B infection.
文摘Hepatitis B virus(HBV) infection is a major global health problem. Although current therapies, such as the use of nucleos(t)ide analogs, inhibit HBV replication efficiently, they do not eliminate covalently closed circular DNA(ccc DNA), which persists in hepatocyte nuclei. As HBV ccc DNA is a viral transcription template, novel therapeutic approaches to directly target HBV ccc DNA are necessary to completely eradicate persistent HBV infections. HBV ccc DNA levels in HBV-infected human liver cells are extremely low; thus, more reliable and simple measurement methods are needed to correctly monitor their levels during therapeutic treatment. Although reverse transcription-polymerase chain reaction or Southern blot procedures are currently used in research studies, these methods are not completely reliable and are also time-consuming and labor-intensive. Genome editing technologies, such as zinc finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9(CRISPR/Cas9) system, which are designed to target specific DNA sequences, represent highly promising potential therapeutic tools. In particular, the CRISPR/Cas9 system is an easily customizable sequencespecific nuclease with high flexibility and may be the most feasible approach to target HBV ccc DNA. Further research to develop easier, safer, and more effective protocols should be pursued.
基金Beijing Municipal Science & Technology Commission, No. H020920020690
文摘AIM： To evaluate the effects of antiviral agents and HBV genotypes on intrahepatic covalently closed circular DNA （ccc DNA） in HBeAg-positive chronic hepatitis B patients.METHODS： Seventy-one patients received lamivudine （n = 35）, or sequential therapy with lamivudine- interferon alpha 2b （IFN-α 2b, n = 24） for 48 wk, or IFN-α 2b （n = 12） for 24 wk. All subjects were followed up for 24 wk. Intrahepatic ccc DNA was measured quantitatively by PCR. HBV genotypes were analyzed by PCR-RFLP.RESULTS： Sequential lamivudine- INF-α therapy, lamivudine and INF-α monotherapy reduced ccc DNA of 1.7 log, 1.4 log and 0.8 log, respectively （P 〈 0.05）. Seventeen out of the 71 patieots developed HBeAg seroconversion, the reduction of ccc DNA in the HBeAg seroconversion patients was more significant than that in the HBeAg positive patients （3.0 log vs 1.6 log, P = 0.0407）. Twenty-four weeks after antiviral therapy withdrawal, 16 patients had a sustained virological response, the baseline intrahepatic ccc DNA in the patients with a sustained virological response was significantly lower than that in the patients with virological rebound （4.6 log vs 5.4 log, P = 0.0472）. HBV genotype C accounted for 85.9% （n = 61）, and genotype B for 14.1% （n = 10）, respectively, in the 71 patients. There was no significant difference in the change of ccc DNA level between HBV genotypes C and B （2.1 log vs 1.9 log）.CONCLUSION： Forty-eight week sequential lamivudine- INF-α therapy and lamivudine monotherapy reduce ccc DNA more significantly than 24-wk INF-α monotherapy. Low baseline intrahepatic ccc DNA level may predict the long-term efficacy of antiviral treatment. HBV genotypes C and B have no obvious influence on ccc DNA load.
文摘250 million people worldwide continue to be chronically infected with the virus.While patients may be treated with nucleoside/nucleotide analogues,this only suppresses HBV titre to sub-detection levels without eliminating the persistent HBV covalently closed circular DNA(cccDNA)genome.As a result,HBV infection cannot be cured,and the virus reactivates when conditions are favorable.Interferons(IFNs)are cytokines known to induce powerful antiviral mechanisms that clear viruses from infected cells.They have been shown to induce cccDNA clearance,but their use in the treatment of HBV infection is limited as HBVtargeting immune cells are exhausted and HBV has evolved multiple mechanisms to evade and suppress IFN signalling.Thus,to fully utilize IFN-mediated intracellular mechanisms to effectively eliminate HBV,instead of direct IFN administration,novel strategies to sustain IFN-mediated anti-cccDNA and antiviral mechanisms need to be developed.This review will consolidate what is known about how IFNs act to achieve its intracellular antiviral effects and highlight the critical interferon-stimulated gene targets and effector mechanisms with potent anti-cccDNA functions.These include cccDNA degradation by APOBECs and cccDNA silencing and transcription repression by epigenetic modifications.In addition,the mechanisms that HBV employs to disrupt IFN signalling will be discussed.Drugs that have been developed or are in the pipeline for components of the IFN signalling pathway and HBV targets that detract IFN signalling mechanisms will also be identified and discussed for utility in the treatment of HBV infections.Together,these will provide useful insights into design strategies that specifically target cccDNA for the eradication of HBV.
基金Supported by International Cooperation 2021 with Indonesia from the Regione of Friuli Venezia Giulia(Prot.0015911/P)to the FIF.
文摘Chronic infection with hepatitis B virus(HBV)remains a major global health problem,especially in developing countries.It may lead to prolonged liver damage,fibrosis,cirrhosis,and hepatocellular carcinoma.Persistent chronic HBV infection is related to host immune response and the stability of the covalently closed circular DNA(cccDNA)in human hepatocytes.In addition to being essential for viral transcription and replication,cccDNA is also suspected to play a role in persistent HBV infections or hepatitis relapses since cccDNA is very stable in non-dividing human hepatocytes.Understanding the pathogenicity and oncogenicity of HBV components would be essential in the development of new diagnostic tools and treatment strategies.This review summarizes the role and molecular mechanisms of HBV cccDNA in hepatocyte transformation and hepatocarcinogenesis and current efforts to its detection and targeting.
文摘Binding of cordycepin to the double helical DNA with a high affinity was investigated by CD spectra in this paper. The results proved that uncoiling, unbinding and denaturation of DNA proceeded continuously upon the increase of the concentration of cordycepin.
基金Supported by the Henan Medical Science and Technology Research Program,No.LHGJ20210045.
文摘BACKGROUND Minimally invasive or noninvasive,sensitive and accurate detection of colorectal cancer(CRC)is urgently needed in clinical practice.AIM To identify a noninvasive,sensitive and accurate circular free DNA marker detected by digital polymerase chain reaction(dPCR)for the early diagnosis of clinical CRC.METHODS A total of 195 healthy control(HC)individuals and 101 CRC patients(38 in the early CRC group and 63 in the advanced CRC group)were enrolled to establish the diagnostic model.In addition,100 HC individuals and 62 patients with CRC(30 early CRC and 32 advanced CRC groups)were included separately to validate the model.CAMK1D was dPCR.Binary logistic regression analysis was used to establish a diagnostic model including CAMK1D and CEA.RESULTS To differentiate between the 195 HCs and 101 CRC patients(38 early CRC and 63 advanced CRC patients),the common biomarkers CEA and CAMK1D were used alone or in combination to evaluate their diagnostic value.The area under the curves(AUCs)of CEA and CAMK1D were 0.773(0.711,0.834)and 0.935(0.907,0.964),respectively.When CEA and CAMK1D were analyzed together,the AUC was 0.964(0.945,0.982).In differentiating between the HC and early CRC groups,the AUC was 0.978(0.960,0.995),and the sensitivity and specificity were 88.90%and 90.80%,respectively.In differentiating between the HC and advanced CRC groups,the AUC was 0.956(0.930,0.981),and the sensitivity and specificity were 81.30%and 95.90%,respectively.After building the diagnostic model containing CEA and CAMK1D,the AUC of the CEA and CAMK1D joint model was 0.906(0.858,0.954)for the validation group.In differentiating between the HC and early CRC groups,the AUC was 0.909(0.844,0.973),and the sensitivity and specificity were 93.00%and 83.30%,respectively.In differentiating between the HC and advanced CRC groups,the AUC was 0.904(0.849,0.959),and the sensitivity and specificity were 93.00%and 75.00%,respectively.CONCLUSION We built a diagnostic model including CEA and CAMK1D for differentiating between HC individuals and CRC patients.Compared with the common biomarker CEA alone,the diagnostic model exhibited significant improvement.
基金supported by the National Key Research and Development Program of China(2016YFD0500705)Central Public-interest Scientific Institution Basal Research Fund(No.1610302019002)
文摘Fur seal feces-associated circular DNA virus(FSfa CV)is an unclassified circular replication-associated protein(Rep)-encoding single-stranded(CRESS)DNA virus that has been detected in mammals(fur seals and pigs).The biology and epidemiology of the virus remain largely unknown.To investigate the virus diversity among pigs in Anhui Province,China,we pooled 600 nasal samples in 2017 and detected viruses using viral metagenomic methods.From the assembled contigs,12 showed notably high nucleotide acid sequence similarities to the genome sequences of FSfa CVs.Based on these sequences,a full-length genome sequence of the virus was then obtained using overlapping PCR and sequencing,and the virus was designated as FSfa CV-CHN(Gen Bank No.MK462122).This virus shared 91.3%and 90.9%genome-wide nucleotide sequence similarities with the New Zealand fur seal strain FSfa CV-as50 and the Japanese pig strain FSfa CVJPN1,respectively.It also clustered with the two previously identified FSfa CVs in a unique branch in the phylogenetic tree based on the open reading frame 2(ORF2),Rep-coding gene,and the genome of the reference CRESS DNA viruses.Further epidemiological investigation using samples collected in 2018 showed that the overall positive rate for the virus was 56.4%(111/197)in Anhui Province.This is the first report of FSfa CVs identified in pigs in China,and further epidemiological studies are warranted to evaluate the influence of the virus on pigs.
基金Supported by National Natural Science Foundation of China,No. 81972010National Key Research and Development Program of China,No. 2020YFC2002700National Key Research and Development Program of China,No. 2020YFC2004604。
文摘BACKGROUND Colorectal cancer(CRC) is the third most common cancer worldwide, and it is the second leading cause of death from cancer in the world, accounting for approximately 9% of all cancer deaths. Early detection of CRC is urgently needed in clinical practice.AIM To build a multi-parameter diagnostic model for early detection of CRC.METHODS Total 59 colorectal polyps(CRP) groups, and 101 CRC patients(38 early-stage CRC and 63 advanced CRC) for model establishment. In addition, 30 CRP groups,and 62 CRC patients(30 early-stage CRC and 32 advanced CRC) were separately included to validate the model. 51 commonly used clinical detection indicators and the 4 extrachromosomal circular DNA markers NDUFB7, CAMK1D, PIK3CD and PSEN2 that we screened earlier. Four multi-parameter joint analysis methods:binary logistic regression analysis, discriminant analysis, classification tree and neural network to establish a multi-parameter joint diagnosis model.RESULTS Neural network included carcinoembryonic antigen(CEA), ischemia-modified albumin(IMA),sialic acid(SA), PIK3CD and lipoprotein a(LPa) was chosen as the optimal multi-parameter combined auxiliary diagnosis model to distinguish CRP and CRC group, when it differentiated 59CRP and 101 CRC, its overall accuracy was 90.8%, its area under the curve(AUC) was 0.959(0.934,0.985), and the sensitivity and specificity were 91.5% and 82.2%, respectively. After validation,when distinguishing based on 30 CRP and 62 CRC patients, the AUC was 0.965(0.930-1.000), and its sensitivity and specificity were 66.1% and 70.0%. When distinguishing based on 30 CRP and 32early-stage CRC patients, the AUC was 0.960(0.916-1.000), with a sensitivity and specificity of 87.5% and 90.0%, distinguishing based on 30 CRP and 30 advanced CRC patients, the AUC was 0.970(0.936-1.000), with a sensitivity and specificity of 96.7% and 86.7%.CONCLUSION We built a multi-parameter neural network diagnostic model included CEA, IMA, SA, PIK3CD and LPa for early detection of CRC, compared to the conventional CEA, it showed significant improvement.
基金The National Natural Science Foundation of China under Grant Nos.11464004,11864006 and 11564015Guizhou Scientific and Technological Program(20185781 and 20185781-15)the Foundation of Key Laboratory of Animal Genetics,Breeding and Reproduction in The Plateau Mountainous Region,Ministry of Education(Guizhou University),KY2020243。
文摘Knots are discovered in biophysical systems,such as DNA and proteins.Knotted portions in knotted DNA are significantly bent and their corresponding bending angles are comparable with or larger than the sharp bending angle resulting in flexible defects.The role of flexible defects in the interplay of supercoiling and knotting of circular DNA were predicted by a Monte Carlo simulation.In knotted DNA with a particular knot type,a flexible defect noticeably enhances the supercoiling of the knotted DNA and the decreasing excitation energy makes the knotted portion more compact.A reduction in twist rigidity and unwinding of flexible defects are incorporated into the numerical simulations,so that interplay of supercoiling and knotting of circular DNA is studied under torsional conditions.Increasing unwinding not only results in a wider linking number distribution,but also leads to a drift of the distribution to lower values.A flexible defect has obvious effects on knotting probability.The summation of equilibrium distribution probability for nontrivial knotted DNA with different contour length does not change with excitation energy monotonically and has a maximum at an intermediate value of excitation energy around 5 kBT.In the phase space of knot length and gyration radius of knotted DNA,knot length does not anticorrelate with its gyration radius,which is attributed to the flexible defect in the knotted portion,which leads to the release of bending energy and inhibited the competition between entropy and bending energy.
文摘To develop a fluorescent quantitative PCR assay based on Taq-Man chemistry to detect the covalenfly closed circular DNA （eccDNA） of duck hepatitis B virus （DHBV）, a pair of primers was designed from both sides of the nick in the minus strand of DHBV and a Taq-Man probes between the primers, modified with 6-Fam at 5＇ end and Tamra at its 3＇ end was designed to detect the PCR products during PCR cycles. The DHBV DNA fragment was cloned into vector PUCm-T, and the recombinant plasmid was purified and subsequently qualified as the HBV DNA standard. The experimental conditions and reagents used in PCR assay for amplification were sophisticatedly optimized in order to yield a perfect amplification efficacy and reduce the possibility to produce non-specific amplification. It was demonstrated that the detect limit of assay was 10^3 copies/ml, and a linear standard curve was obtained between 10^5 -10^9 copies/ml [ C1 =-2.8361 ln（x） ＋ 41.45, r =-0.9985]. The coefficient of variation was 0.2%-3.14% and 2.22%-4.43% for intra- and inter-assay respectively. After a dynamic survey on the contents of DHBV DNA in serum of ducks, it was found that its peak value appeared at the second week of birth in ducks. It is evident that this method of Taq-Man fluorescent quantitative PCR assay appears to be simple, sensitive and specific.
文摘AIM: To investigate the reduction in hepatitis B virus(HBV) covalently closed-circular DNA(ccc DNA) with entecavir(ETV) or lamivudine(LAM). METHODS: This analysis included patients who had participated in the randomized Phase Ⅲ study ETV-022 comparing ETV vs LAM in nucleos(t)ide-naive, HBe Agpositive patients. Patients received ETV(0.5 mg daily) or LAM(100 mg daily) for a minimum of 52 wk. Patients were eligible to participate in this sub-study if they had paired biopsies at baseline and week 48 with evaluable measurements for hepatic HBV ccc DNA and total hepatic HBV DNA. The main objective was to compare changes in hepatic HBV ccc DNA and total hepatic HBV DNA at week 48 of ETV or LAM treatment, which was a secondary endpoint of study ETV-022. Additional post hoc analyses included linear regression analyses to assess associations of baseline levels and on-treatment changes of ccc DNA with other baseline factors [sex,age, serum HBV DNA, alanine aminotransferase(ALT), Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBV genotype], or ontreatment factors(changes from baseline at week 48 in serum HBV DNA, ALT, Knodell necroinflammatory score, Ishak fibrosis score, total hepatic HBV DNA, and HBe Ag loss at week 48).RESULTS: Overall, 305 patients(ETV = 159; LAM = 146) of ETV-022 had paired baseline and week 48 liver biopsies with evaluable measurements for hepatic HBV ccc DNA and total hepatic HBV DNA, and were included in this analysis. Baseline demographics and disease characteristics were comparable between the two arms. After 48 wk, ETV resulted in significantly greater reductions in hepatic HBV ccc DNA [-0.9 log10 copies/human genome equivalent(HGEq) vs-0.7 log10 copies/HGEq; P = 0.0033] and total hepatic DNA levels(-2.1 log10 copies/HGEq vs-1.6 log10 copies/HGEq; P < 0.0001) than LAM. Virologic, biochemical, and histologic response rates at week 48 were also greater with ETV than with LAM. Baseline HBV ccc DNA levels were positively associated with baseline levels of serum HBV DNA and total hepatic HBV DNA, and negatively associated with HBV genotype F. On-treatment changes in HBV ccc DNA levels were negatively associated with baseline levels of serum HBV DNA and baseline ALT, and were positively associated with on-treatment changes in the levels of serum HBV DNA, total hepatic HBV DNA levels, and ALT, change in Knodell necroinflammatory score, and HBe Ag loss.CONCLUSION: Forty-eight weeks of ETV resulted in greater reductions in ccc DNA and total hepatic HBV DNA than LAM, but long-term therapy may be needed for ccc DNA elimination.
文摘目的运用meta分析方法系统评价补肾方剂治疗男性不育症患者精子DNA损伤(SDF)的有效性和安全性。方法从中国知网、维普、万方、PubMed、Cochrane Library、Embase、Web of Science数据库中检索补肾方剂改善SDF的随机对照试验(RCT)研究,对文献进行方法学质量评价,运用RevMan5.3.5软件进行meta分析,并对结局指标进行GRADE质量分级。结果共纳入17项RCT研究,涉及2164例男性患者。与西医常规治疗相比,补肾方剂能够显著改善男性不育症患者DNA碎片指数(DFI)(MD=-6.50,95%CI:-7.88~-5.11,P<0.01),提高配偶妊娠率(RR=2.11,95%CI:1.12~4.00,P=0.87)、精子总活率(MD=5.56,95%CI:4.39~6.74,P<0.01)、前向运动精子百分率(MD=6.82,95%CI:5.62~8.03,P<0.01)、精子浓度(MD=6.51,95%CI:3.81~9.21,P<0.01)和正常形态精子百分率(MD=1.26,95%CI:0.45~2.06,P<0.01),且补肾方剂不会增加不良反应的发生。结论低到中等质量证据表明,与西医常规治疗相比,补肾方剂在改善男性不育症患者DFI、精液参数、配偶妊娠率等方面具有一定优势,且安全性较好。
基金Supported by the National Natural Science Foundation of China under Grant Nos.11047022,11204045,and 11464004Guizhou Provincial Tracking Key Program of Social Development(SY20123089,SZ20113069)+2 种基金the General Financial Grant from the China Postdoctoral Science Foundation(2014M562341)the Research Foundation for Young University Teachers from Guizhou University(201311)College Innovation Talent Team of Guizhou Province(2014)32
文摘The different topological states of circular double-stranded DNA can be defined by their linking number. The equilibrium distribution of linking number can be obtained by circularizing a linear DNA into a circle by ligase. Based on the recent experimental results that the DNA bending rigidity and twist rigidity strongly depend on temperature, the reduced bending rigidity can be approximated by g = （3.19 x10-19 - T. 4.14s10-22） erg. cm over the temperature interval （5 ~ 53） ~ C, and the temperature dependence of twist rigidity can be fitted by C （ T） = （4588.89 exp（-T/117.04）- 251.33） nm. The temperature dependence of the linking number distribution of circular DNAs can be predicted by using Monte Carlo simulation. The variance of linking number distribution on temperature is in accordance with the previous experimental results. Compared with the temperature dependence of bending rigidity, the temperature dependence of twist rigidity causes a noticeable fluctuation in linking number distribution and mainly contribute towards the variance change of linking number distribution of circular DNA. The variance of the writhe number and twist number in the equation （（ALk）21 = （（ATw）2） -b （（Wr）2） depends on the length of circular DNA. When the length of circular DNA is less than 230 nm, the variance of twist number （（ATw）2） is dominant over the variance of writhe number （（（wr）2））whereas for the condition that the length of the circular DNA is larger than 370 nm.
文摘Circular dichroism (CD) is a reliable,sensitiveand convenient methods for analysing the effectof drugs on DNA,which intercalated into theDNA molecule or formed DNA complex and couldchange the conformation of DNA and alter theCD spectra.Mutagenicity of 19 drugs
