DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to env...DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.展开更多
In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for ...In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.展开更多
During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we perfo...During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH (genome in situ hybridization) and AFLP (amplified fragment length po-lymorphism) on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary struc-ture variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism (MSAP) analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translo-cation lines (CN12, 20.15%; CN17, 20.91%; CN18, 22.42%), but the ratios of hemimethylated sites were significantly lowered (CN12, 21.41%; CN17, 23.43%; CN18, 22.42%), whereas 16.37% were fully-me-thylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of me-thylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns (33.74%), 9 demethylation patterns (22.76%) and 7 uncertain patterns (4.07%). In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.展开更多
With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a c...With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.展开更多
Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence i...Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence immunoassay system was designed.We encapsulated carbon dots with a high fluorescence quantum yield(QY=92.5%)inside silicon nanocapsules to serve as fluorescent markers.These markers were then integrated with the streptavidin(SA)-biotin biomagnification system and immunomagnetic separation technology for the sensitive detection of GFAP.Based on the signal cascade amplification effect of the silicon nanocapsules and SA-biotin,the fluorescence signal of the SA-biotin-modified immunofluorescence nanocapsules increased 3.6-fold compared to the carbon dot-based immunoprobe.The fluorescence immunoassay system was constructed for GFAP using SA-biotin-modified immunocapsules as the sensing probe and immunomagnetic nanoparticles as the immunorecognition probe.The fluorescence immunoassay system can specifically and ultra-sensitively quantify GFAP in blood samples,with a detection range of 10 pg/mL–10 ng/mL and detection limits of 3.2 pg/mL(serum)and 3.6 pg/mL(plasma).Moreover,the fluorescence immunoassay system exhibited prominent recoveries of 99.4%–100.4%(phosphate buffered saline),96%–102.6%(serum),and 93.2%–110.2%(plasma),with favorable specificity and excellent stabilization.The novel fluorescence immunoassay system provides a new approach to the clinical analysis of GFAP and may serve as a potential tool for screening and diagnosing TBI.展开更多
Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing ...Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing remains challenging.Here,a novel nanozyme-triggered polymerization amplification strategy was proposed for constructing highly sensitive surface plasmon resonance(SPR)immunosensor.In detail,Au@Pd core-shell nanooctahedra nanozyme with superior peroxidase(POD)-like activity was synthesized and utilized as a label probe.Simultaneously,Au@Pd core-shell nanooctahedra nanozyme can catalyze the decomposition of H_(2)O_(2)to form hydroxyl radicals(·OH)that triggers the polymerization of aniline to form polyaniline attaching on the surface of sensor chip,significantly amplifying SPR responses.The sensitivity of SPR immunosensor was enhanced by nanozyme-triggered polymerization amplification strategy.Using human immunoglobulin G(HIgG)as a model,the constructed SPR immunosensor obtains a wide linear range of 0.005–1.0μg/m L with low detection limit of 0.106 ng/m L.This research provides new sights on establishing sensitive SPR immunosensor and may evokes more inspiration for developing signal amplification methods based on nanozyme in biosensing.展开更多
Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitori...Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitoring.Nevertheless,challenges still exist in detecting ultra-low concentration analytes due to the inherent sensitivity limitations of LFIA.Recently,significant advances have been achieved by integrating enzyme activity probes and transforming LFIA into a highly sensitive tool for rapidly detecting trace analyte concentrations.Specifically,modifying natural enzymes or engineered nanozymes allows them to function as immune probes,directly catalyzing the production of signal molecules or indirectly initiating enzyme activity.Therefore,the signal intensity and detection sensitivity of LFIA are markedly elevated.The present review undertakes a comprehensive examination of pertinent research literature,offering a systematic analysis of recently proposed enzyme-based signal amplification strategies.By way of comparative assessment,the merits and demerits of current approaches are delineated,along with the identification of research avenues that still need to be explored.It is anticipated that this critical overview will garner considerable attention within the biomedical and materials science communities,providing valuable direction and insight toward the advancement of high-performance LFIA technologies.展开更多
To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generali...To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generalized nonlinear Schrödinger equation and the rate equations.However,this approach is burdened by substantial computational demands,resulting in significant time expenditures.In the context of intelligent laser optimization and inverse design,the necessity for numerous simulations further exacerbates this issue,highlighting the need for fast and accurate simulation methodologies.Here,we introduce an end-to-end model augmented with active learning(E2E-AL)with decent generalization through different dedicated embedding methods over various parameters.On an identical computational platform,the artificial intelligence–driven model is 2000 times faster than the conventional simulation method.Benefiting from the active learning strategy,the E2E-AL model achieves decent precision with only two-thirds of the training samples compared with the case without such a strategy.Furthermore,we demonstrate a multi-objective inverse design of the CPA systems enabled by the E2E-AL model.The E2E-AL framework manifests the potential of becoming a standard approach for the rapid and accurate modeling of ultrafast lasers and is readily extended to simulate other complex systems.展开更多
DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide ...DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.展开更多
Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effective...Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effectiveness once BB has spread. Consequently, it is imperative to develop a rapid, highly sensitive, specific, and easy-to-use detection technique for early on-site diagnosis of BB. We first developed a recombinase-aided amplification-lateral flow dipstick(RAA-LFD) technique for the on-site detection of Xoo. The optimized reaction temperature and time were 37 ℃ and 20 min, indicating that the reaction system can be initiated by body temperature independently of any precision instruments. Evaluation of the RAA-LFD technique using the primers(RAAF2/R2) and probe(RAA2-nfo-probe) derived from the Xoo ORF0080 locus exhibited high specificity and eliminated cross-reactivity with other bacterial species. The sensitivity of RAA-LFD is up to 1 pg/μL for Xoo genomic DNA and 100 CFU/m L for Xoo cells. Significantly, this technique accurately detected Xoo from both artificially inoculated and naturally infected rice leaves at the early stage of infection, directly deploying plant tissue fluid as the template without DNA extraction. These attributes make the developed RAA-LFD system a viable technique for the early diagnosis of BB in the field, providing technical support for early-warning systems and disease control.展开更多
Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,...Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,we reported a liposome-mediated signal-off photoelectrochemical(PEC)immunoassay for the sensitive detection of carcinoembryonic antigen(CEA)using ternary transition metal sulfide CuS/ZnCdS as the photoactive material.Good photocurrents were acquired on the basis of specific oxidation reaction of dopamine on the CuS/ZnCdS.The energy band relationship of CuS/ZnCdS was determined,and the wellmatched oxidation potential of dopamine was verified.To achieve accurate recovery of low-abundance CEA,systematic PEC evaluation from human serum samples was performed by combining with classical immunoreaction and liposome-induced dopamine amplification strategy with high stability and selectivity.Under optimum conditions,PEC immunoassay displayed good photocurrent responses toward target CEA with a dynamic linear range of 0.1-50 ng/mL with a detection limit of 31.6 pg/mL.Importantly,this system by combining with a discussion of energy level matching between semiconductor energy bands and small-molecules opens a new horizon for development of high-efficient PEC immunoassays.展开更多
Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an a...Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an asymmetric triangular hill on Earth and SH waves.Firstly,based on the development of wave functions and regional matching techniques,we introduce a semi-circular artificial auxiliary boundary,dividing the solution model into a semi-infinite body containing a semi-circular depression and an asymmetric fan-shaped region.Secondly,we derive the domain function form applicable to solving asymmetric problems.Utilizing the theory of complex variables,we establish a well-posed matrix for solving domain functions within the same coordinate system.Numerical results demonstrate that the scattering of SH waves by a protuberance is jointly influenced by the geometric parameters of the hill and the angle of incidence.Additionally,the frequency of the incident wave also has a certain degree of impact on the displacement amplitude.This study elucidates the scattering mechanism of SH waves by complex boundaries,providing a theoretical reference for building site selection and seismic design.In practical problems,the asymmetric assumption is more applicable than the symmetry assumption.展开更多
Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly l...Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly limited its therapeutic effect.Herein,we prepared a pH-responsiveβ-lapachone-loaded ironpolyphenol nanocomplex(LIPN)through a one-pot method.β-Lapachone in LIPN selectively enhanced H_(2)O_(2)concentration in tumor cells,and ferrous ions cascadely generated abundant cytotoxic•OH.Therefore,LIPN with cascade amplification of reactive oxygen species(ROS)showed high chemodynamic cytotoxicity in tumor cells,efficiently improving the expression of damage-associated molecular patterns(DAMPs),and exerting strong immunogenic cell death(ICD).As a result,LIPN exhibited efficient tumor inhibition ability in 4T1 subcutaneous tumor model in vivo with great biocompatibility.Additionally,the infiltration of cytotoxic CD8^(+)T lymphocytes and inhibition of regulatory CD4^(+)FoxP3^(+)T lymphocytes in tumors demonstrated the activation of immunosuppressive tumor microenvironment by LIPN-induced ICD.Therefore,this work provided a new approach to enhance ICD of chemodynamic therapy through selective cascade amplification of ROS in cancer cells.展开更多
Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein ef...Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein effectors,enables a new amplification-free nucleic acid diagnostic tool.In this study,we develop a CRISPR-graphene field-effect transistors(GFETs)biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection.This biosensor exploits the target RNA-activated continuous ss DNA cleavage activity of the d Csm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free,amplification-free,highly sensitive,and specific RNA detection.The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs,with detection limits at the aM level and a broad linear range of 10^(-15)to 10^(-11)M for RNAs and 10^(-15)to 10^(-9)M for miRNAs.It shows high sensitivity in throat swabs and serum samples,distinguishing between healthy individuals(N=5)and breast cancer patients(N=6)without the need for extraction,purification,or amplification.This platform mitigates risks associated with nucleic acid amplification and cross-contamination,making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.展开更多
Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison e...Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).展开更多
[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 su...[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.展开更多
[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional tou...[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.展开更多
Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set...Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.展开更多
A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encod...A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.展开更多
It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic la...It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic layer of maize seeds.Tyramide signal amplification fluorescence in situ hybridization (TSA FISH) is a novel and high sensitive FISH technique,which is suitable for routine application in plant cytogenetic research.Using this technique,we physically mapped the bz1 gene onto the short arm of chromosome 9 and the long arm of chromosome 1;the percentage distances from centromere to hybridization site were 40.2,75.4 respectively,and the bz2 onto the long arm of chromosome 1 and the short arm of chromosome 5;the percentage distances from centromere to hybridization site were 21.6,15.3 separately.The TSA FISH techniques of small low copy DNA sequences for plants are discussed.展开更多
基金supported by National Nonprofit Institute Research Grant of Chinese Academy of Forestry(TGB2013010)
文摘DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.
基金supported by the grants from the National Natural Science Foundation of China (No. 4147 6124)the Natural Science Foundation of Zhejiang Province (No. LY17C190005)+3 种基金the Major Agriculture Program of Ningbo (No. 2017C110007)the Ningbo Science and Technology Project (No. 2016C10037)the Open Fund of Ningbo University (No. xkzsc1505)K C Wong Magana Fund in Ningbo University
文摘In this study,the methylation-sensitive amplification polymorphism(MSAP)was used to compare the genomic DNA methylation level of muscle,gill and hepatopancreas of Portunus trituberculatus subjected to salinity 12 for 30 days to illustrate the epigenetic mechanism of osmoregulation.Thirty primers were used to analyze the difference of methylation level of different tissues.The results showed that the baseline methylation level of muscle,hepatopancreas and gill was 47.31%,22.94%and 17.69%,respectively.After exposed to low salinity stress,the methylation epiloci changed in the three tissues.Both demethylation and methylation processes occurred under low salinity stress.The methylation ratio decreased in muscle and gill but increased in hepatopancreas.These results indicated that DNA methylation is tissue-specific when P.trituberculatus responds to low salinity.
基金the National Natural Science Foundation of China (Grants No. 30671136 and 30730065)the China Postdoctoral Science Foundation (Grant No. 20070411158)+1 种基金the Program for New Century Excellent Talents in University from Ministry of Education,China (Grant No. NCET-06-0810)the Youth Foundation of University of Electronic Science and Technology of China (Grant No. L08010901JX0677)
文摘During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH (genome in situ hybridization) and AFLP (amplified fragment length po-lymorphism) on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary struc-ture variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism (MSAP) analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translo-cation lines (CN12, 20.15%; CN17, 20.91%; CN18, 22.42%), but the ratios of hemimethylated sites were significantly lowered (CN12, 21.41%; CN17, 23.43%; CN18, 22.42%), whereas 16.37% were fully-me-thylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of me-thylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns (33.74%), 9 demethylation patterns (22.76%) and 7 uncertain patterns (4.07%). In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195)。
文摘With the approval of more and more genetically modified(GM)crops in our country,GM safety management has become more important.Transgenic detection is a major approach for transgenic safety management.Nevertheless,a convenient and visual technique with low equipment requirements and high sensitivity for the field detection of GM plants is still lacking.On the basis of the existing recombinase polymerase amplification(RPA)technique,we developed a multiplex RPA(multi-RPA)method that can simultaneously detect three transgenic elements,including the cauliflower mosaic virus 35S gene(CaMV35S)promoter,neomycin phosphotransferaseⅡgene(NptⅡ)and hygromycin B phosphotransferase gene(Hyg),thus improving the detection rate.Moreover,we coupled this multi-RPA technique with the CRISPR/Cas12a reporter system,which enabled the detection results to be clearly observed by naked eyes under ultraviolet(UV)light(254 nm;which could be achieved by a portable UV flashlight),therefore establishing a multi-RPA visual detection technique.Compared with the traditional test strip detection method,this multi-RPA-CRISPR/Cas12a technique has the higher specificity,higher sensitivity,wider application range and lower cost.Compared with other polymerase chain reaction(PCR)techniques,it also has the advantages of low equipment requirements and visualization,making it a potentially feasible method for the field detection of GM plants.
基金supported by the AMS Funding Project(No.ZZB2023C7010).
文摘Glial fibrillary acidic protein(GFAP)is one of the discriminative biomarkers for diagnosing traumatic brain injury(TBI),and accurate determination of GFAP is clinically significant.In this study,a novel fluorescence immunoassay system was designed.We encapsulated carbon dots with a high fluorescence quantum yield(QY=92.5%)inside silicon nanocapsules to serve as fluorescent markers.These markers were then integrated with the streptavidin(SA)-biotin biomagnification system and immunomagnetic separation technology for the sensitive detection of GFAP.Based on the signal cascade amplification effect of the silicon nanocapsules and SA-biotin,the fluorescence signal of the SA-biotin-modified immunofluorescence nanocapsules increased 3.6-fold compared to the carbon dot-based immunoprobe.The fluorescence immunoassay system was constructed for GFAP using SA-biotin-modified immunocapsules as the sensing probe and immunomagnetic nanoparticles as the immunorecognition probe.The fluorescence immunoassay system can specifically and ultra-sensitively quantify GFAP in blood samples,with a detection range of 10 pg/mL–10 ng/mL and detection limits of 3.2 pg/mL(serum)and 3.6 pg/mL(plasma).Moreover,the fluorescence immunoassay system exhibited prominent recoveries of 99.4%–100.4%(phosphate buffered saline),96%–102.6%(serum),and 93.2%–110.2%(plasma),with favorable specificity and excellent stabilization.The novel fluorescence immunoassay system provides a new approach to the clinical analysis of GFAP and may serve as a potential tool for screening and diagnosing TBI.
基金supported by National Natural Science Foundation of China(Nos.22474124,21575125)the National Natural Science Foundation of Jiangsu Province(No.BK20221370)+4 种基金Key University Natural Science Foundation of Jiangsu-Province(No.20KJA150004)the Project for Science and Technology of Yangzhou(No.YZ2022074)Project for Yangzhou City and Yangzhou University corporation(No.YZ2023204)the Open Research Fund of State Key Laboratory of Analytical Chemistry for Life Science(No.SKLACLS2405)Postgraduate Research&Practice Innovation Program of Jiangsu Province(No.KYCX22_3462)。
文摘Although diverse signal-amplified methods have been committed to improve the sensitivity of surface plasmon resonance(SPR)biosensing,introducing convenient and robust signal amplification strategy into SPR biosensing remains challenging.Here,a novel nanozyme-triggered polymerization amplification strategy was proposed for constructing highly sensitive surface plasmon resonance(SPR)immunosensor.In detail,Au@Pd core-shell nanooctahedra nanozyme with superior peroxidase(POD)-like activity was synthesized and utilized as a label probe.Simultaneously,Au@Pd core-shell nanooctahedra nanozyme can catalyze the decomposition of H_(2)O_(2)to form hydroxyl radicals(·OH)that triggers the polymerization of aniline to form polyaniline attaching on the surface of sensor chip,significantly amplifying SPR responses.The sensitivity of SPR immunosensor was enhanced by nanozyme-triggered polymerization amplification strategy.Using human immunoglobulin G(HIgG)as a model,the constructed SPR immunosensor obtains a wide linear range of 0.005–1.0μg/m L with low detection limit of 0.106 ng/m L.This research provides new sights on establishing sensitive SPR immunosensor and may evokes more inspiration for developing signal amplification methods based on nanozyme in biosensing.
基金Financial supports from the National Natural Science Foundation of China(NSFC,Nos.52272144 and 22205048)Heilongjiang Provincial Natural Science Foundation of China(No.JQ2022E001)+3 种基金China Postdoctoral Science Foundation(Nos.2022M710931 and 2023T160154)Heilongjiang Postdoctoral Science Foundation(No.LBH-Z22010)Natural Science Foundation of Shandong Province(No.ZR2020ZD42)the Fundamental Research funds for the Central Universities are greatly acknowledged.
文摘Lateral flow immunoassay(LFIA),a rapid detection technique noted for simplicity and economy,has showcased indispensable applicability in diverse domains such as disease screening,food safety,and environmental monitoring.Nevertheless,challenges still exist in detecting ultra-low concentration analytes due to the inherent sensitivity limitations of LFIA.Recently,significant advances have been achieved by integrating enzyme activity probes and transforming LFIA into a highly sensitive tool for rapidly detecting trace analyte concentrations.Specifically,modifying natural enzymes or engineered nanozymes allows them to function as immune probes,directly catalyzing the production of signal molecules or indirectly initiating enzyme activity.Therefore,the signal intensity and detection sensitivity of LFIA are markedly elevated.The present review undertakes a comprehensive examination of pertinent research literature,offering a systematic analysis of recently proposed enzyme-based signal amplification strategies.By way of comparative assessment,the merits and demerits of current approaches are delineated,along with the identification of research avenues that still need to be explored.It is anticipated that this critical overview will garner considerable attention within the biomedical and materials science communities,providing valuable direction and insight toward the advancement of high-performance LFIA technologies.
基金supported by the National Natural Science Foundation of China(Grant Nos.62227821,62025503,and 62205199).
文摘To capture the nonlinear dynamics and gain evolution in chirped pulse amplification(CPA)systems,the split-step Fourier method and the fourth-order Runge–Kutta method are integrated to iteratively address the generalized nonlinear Schrödinger equation and the rate equations.However,this approach is burdened by substantial computational demands,resulting in significant time expenditures.In the context of intelligent laser optimization and inverse design,the necessity for numerous simulations further exacerbates this issue,highlighting the need for fast and accurate simulation methodologies.Here,we introduce an end-to-end model augmented with active learning(E2E-AL)with decent generalization through different dedicated embedding methods over various parameters.On an identical computational platform,the artificial intelligence–driven model is 2000 times faster than the conventional simulation method.Benefiting from the active learning strategy,the E2E-AL model achieves decent precision with only two-thirds of the training samples compared with the case without such a strategy.Furthermore,we demonstrate a multi-objective inverse design of the CPA systems enabled by the E2E-AL model.The E2E-AL framework manifests the potential of becoming a standard approach for the rapid and accurate modeling of ultrafast lasers and is readily extended to simulate other complex systems.
基金supported by the National Natural Science Foundation of China(Nos.21874060 and 22174058,U21A20282)the Science and Technology program of Gansu Province(No.22JR5RA476)。
文摘DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability,and their abnormal expression induced various human cancers.Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme.Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes,the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores.Thus,it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection.Herein,we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1(FEN1),human alkyladenine DNA glycosylase(hAAG)and uracil DNA glycosylase(UDG)based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence(CE-LIF)detection.The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation.The method demonstrated a detection limit of 0.07 U/mL(0.08-160 U/mL)for FEN1,2.40 U/mL(2.5-250U/mL)for hAAG and 2.1×10^(-4)U/mL(0.0004-2.5 U/mL)for UDG,the relative standard deviations(RSDs)of peak time and peak area for different analytes were as follows:2.50%-4,37%and 3.24%-7.18%(inter-day);1.37%-2.71%and 1.43%-3.02%(intra-day),4.28%-6.08%and 4.16%-7.57%(column to column),respectively.And it can identify the inhibitor-like drugs,evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts,providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.
基金supported by the Zhejiang Provincial Natural Science Foundation of China(Grant Nos.LY23C130004 and LZ24C130004)the National Natural Science Foundation of China(Grant No.32472115)+1 种基金the National Key Research and Development Program of China(Grant No.2022YFF1003301)the Agricultural Sciences and Technologies Innovation Program of Chinese Academy of Agricultural Sciences。
文摘Bacterial blight(BB) is a devastating worldwide rice disease caused by Xanthomonas oryzae pv. oryzae(Xoo), which is difficult to diagnose based on early symptoms. Conventional chemical control yields limited effectiveness once BB has spread. Consequently, it is imperative to develop a rapid, highly sensitive, specific, and easy-to-use detection technique for early on-site diagnosis of BB. We first developed a recombinase-aided amplification-lateral flow dipstick(RAA-LFD) technique for the on-site detection of Xoo. The optimized reaction temperature and time were 37 ℃ and 20 min, indicating that the reaction system can be initiated by body temperature independently of any precision instruments. Evaluation of the RAA-LFD technique using the primers(RAAF2/R2) and probe(RAA2-nfo-probe) derived from the Xoo ORF0080 locus exhibited high specificity and eliminated cross-reactivity with other bacterial species. The sensitivity of RAA-LFD is up to 1 pg/μL for Xoo genomic DNA and 100 CFU/m L for Xoo cells. Significantly, this technique accurately detected Xoo from both artificially inoculated and naturally infected rice leaves at the early stage of infection, directly deploying plant tissue fluid as the template without DNA extraction. These attributes make the developed RAA-LFD system a viable technique for the early diagnosis of BB in the field, providing technical support for early-warning systems and disease control.
基金financial support from the National Natural Science Foundation of China(Nos.22274022 and 21874022).
文摘Development of accurate analytical protocols for cancer biomarkers is used for the initial prescreening of malignant tumors,disease surveillance,and efficacy assessment with significant clinical benefits.In this work,we reported a liposome-mediated signal-off photoelectrochemical(PEC)immunoassay for the sensitive detection of carcinoembryonic antigen(CEA)using ternary transition metal sulfide CuS/ZnCdS as the photoactive material.Good photocurrents were acquired on the basis of specific oxidation reaction of dopamine on the CuS/ZnCdS.The energy band relationship of CuS/ZnCdS was determined,and the wellmatched oxidation potential of dopamine was verified.To achieve accurate recovery of low-abundance CEA,systematic PEC evaluation from human serum samples was performed by combining with classical immunoreaction and liposome-induced dopamine amplification strategy with high stability and selectivity.Under optimum conditions,PEC immunoassay displayed good photocurrent responses toward target CEA with a dynamic linear range of 0.1-50 ng/mL with a detection limit of 31.6 pg/mL.Importantly,this system by combining with a discussion of energy level matching between semiconductor energy bands and small-molecules opens a new horizon for development of high-efficient PEC immunoassays.
基金supported by the National Key R&D Program of China(Grant No.2022YFC3003601)Joint Funds of the National Natural Science Foundation of China Project on Earthquake Science(Grant No.U2239252)the program of the Innovative Research Team in China Earthquake Administration.
文摘Surface irregularities,such as hills and ridges,can significantly amplify ground motion caused by earthquakes.Therefore,in this study,we propose an analytical solution model to investigate the interaction between an asymmetric triangular hill on Earth and SH waves.Firstly,based on the development of wave functions and regional matching techniques,we introduce a semi-circular artificial auxiliary boundary,dividing the solution model into a semi-infinite body containing a semi-circular depression and an asymmetric fan-shaped region.Secondly,we derive the domain function form applicable to solving asymmetric problems.Utilizing the theory of complex variables,we establish a well-posed matrix for solving domain functions within the same coordinate system.Numerical results demonstrate that the scattering of SH waves by a protuberance is jointly influenced by the geometric parameters of the hill and the angle of incidence.Additionally,the frequency of the incident wave also has a certain degree of impact on the displacement amplitude.This study elucidates the scattering mechanism of SH waves by complex boundaries,providing a theoretical reference for building site selection and seismic design.In practical problems,the asymmetric assumption is more applicable than the symmetry assumption.
基金supported by the National Natural Science Foundation of China(Nos.T2293753,52203194)the National Key R&D Program of China(No.2021YFA1201200)+1 种基金the Natural Science Foundation of Zhejiang Province(No.LR18E030002)2023 Hangzhou West Lake Pearl Project Leading Innovative Youth Team Project.
文摘Chemodynamic therapy(CDT),using Fenton agents to generate highly cytotoxic•OH from H_(2)O_(2)has been demonstrated as a powerful anticancer method.However,the insufficient endogenous H_(2)O_(2)in tumor cells greatly limited its therapeutic effect.Herein,we prepared a pH-responsiveβ-lapachone-loaded ironpolyphenol nanocomplex(LIPN)through a one-pot method.β-Lapachone in LIPN selectively enhanced H_(2)O_(2)concentration in tumor cells,and ferrous ions cascadely generated abundant cytotoxic•OH.Therefore,LIPN with cascade amplification of reactive oxygen species(ROS)showed high chemodynamic cytotoxicity in tumor cells,efficiently improving the expression of damage-associated molecular patterns(DAMPs),and exerting strong immunogenic cell death(ICD).As a result,LIPN exhibited efficient tumor inhibition ability in 4T1 subcutaneous tumor model in vivo with great biocompatibility.Additionally,the infiltration of cytotoxic CD8^(+)T lymphocytes and inhibition of regulatory CD4^(+)FoxP3^(+)T lymphocytes in tumors demonstrated the activation of immunosuppressive tumor microenvironment by LIPN-induced ICD.Therefore,this work provided a new approach to enhance ICD of chemodynamic therapy through selective cascade amplification of ROS in cancer cells.
基金financially supported by the National Science and Technology Innovation 2030 Grants(2021ZD0201600)the National Key R&D Program of China(2021YFA0717000)+2 种基金the Intramural Joint Program Fund of State Key Laboratory of Microbial Technology(Project No.SKLMTIJP-2024-05)the Natural Science Foundation of Qingdao-Original exploration project(Project No.24-4-4-zrjj-139-jch)the National Natural Science Foundation of China(31771380)。
文摘Amplification-free,highly sensitive,and specific nucleic acid detection is crucial for health monitoring and diagnosis.The type III CRISPR-Cas10 system,which provides viral immunity through CRISPRassociated protein effectors,enables a new amplification-free nucleic acid diagnostic tool.In this study,we develop a CRISPR-graphene field-effect transistors(GFETs)biosensor by combining the type III CRISPR-Cas10 system with GFETs for direct nucleic acid detection.This biosensor exploits the target RNA-activated continuous ss DNA cleavage activity of the d Csm3 CRISPR-Cas10 effector and the high charge density of a hairpin DNA reporter on the GFET channel to achieve label-free,amplification-free,highly sensitive,and specific RNA detection.The CRISPR-GFET biosensor exhibits excellent performance in detecting medium-length RNAs and miRNAs,with detection limits at the aM level and a broad linear range of 10^(-15)to 10^(-11)M for RNAs and 10^(-15)to 10^(-9)M for miRNAs.It shows high sensitivity in throat swabs and serum samples,distinguishing between healthy individuals(N=5)and breast cancer patients(N=6)without the need for extraction,purification,or amplification.This platform mitigates risks associated with nucleic acid amplification and cross-contamination,making it a versatile and scalable diagnostic tool for molecular diagnostics in human health.
基金supported by the University-Industry Col aborative Education Program,China(220904860093831)。
文摘Porcine Contagious Pleuropneumonia(PCP)is a respiratory infectious disease of pigs caused by Actinobacillus pleuropneumoniae.The disease has been prevalent in pig farms since it was first identified in 1957(Pattison et al.1957).
基金Supported by Important Project of Jinlin Provincial Science and Technology Department(20065020)~~
文摘[Objective] The research aimed to design primers that are suitable for detecting H5 and H7 subtypes of avian influenza virus （AIV） ; [Method] DNAStar was used to analyze the homology of the sequences of H5 and H7 subtypes of AIV accessed in GenBank, and design primers（ by Primer Premier 5.0） on high homologous region of these sequences, and then amplified by RT-PCR. [Result] The multiplex RT-PCR amplification, agarose gel electrophoresis and sequencing results showed that the self-designed primers are successful for detecting AIV. [Conclusion] It is feasible to rapidly diagnose AIV through this method.
文摘[Objective] The aim was to explore the special methods for amplification of large-family genes by using primers with high degeneracy.[Method] By using the primers with high degeneracy,conventional PCR,conventional touchdown PCR and the optimized abnormal touchdown PCR were respectively carried out to amplify the genomic DNA of Cyprinus carpio.[Result] Only one evident electrophoretic band and a few Sox genes were obtained by using normal PCR;no obvious electrophoretic band but dispersive product was obtained by normal touchdown PCR;ideal result was obtained by the abnormal touchdown PCR that three evident electrophoretic bands and much more Sox genes were amplified.[Conclusion] The research provided theoretical basis for the optimization and selection of PCR amplification conditions of the large-family genes.
文摘Objective] This study aimed to develop a reverse transcription loop-medi-ated isothermal amplification （RT-LAMP） method for detecting BVDV. [Method] Since gp48 gene of BVDV is among the most conserved regions, a set of four primers was designed to amplify six target sequences at the gp48 gene region for the RT-LAMP assay. The optimization of the RT-LAMP reaction was performed by evaluat-ing reaction temperature and reaction time. [Result] The RT-LAMP aasay was suc-cessful y conducted at 56 ℃ within 40 min under isothermal conditions, and the re-sults could be detected as ladder-like bands using agarose gel electrophoresis. The RT-LAMP assay is highly sensitive and able to detect 3.74 ×100 copies/μl of BVDV RNA, as no cross-reaction was observed with other viruses. [Conclusion] Overal , the newly established RT-LAMP assay indicates the potential application in both clinical diagnosis and field surveil ance of BVDV.
文摘A reverse-transcription loop-mediated isothermal amplification （RT-LAMP） method was established for the detection of wheat streak mosaic virus （WSMV）. Ac-cording to the conservative regions of the genes that encode the coat protein of WSMV, 2 pairs of primers were designed. Final y, the 1st pair of primers was select-ed through the specificity test. The sensitivity test showed the sensitivity of RT-LAMP method was 10 times higher than that of RT-PCR. In addition, the amplifica-tion of target gene could be judged visual y from the presence of fluorescence （cal-cein） in the final reaction system. The RT-LAMP method, established in this study, was rapid, easy, specific and sensitive. Moreover, it did not require sophisticated equip-ment. The RT-LAMP was suitable for the rapid detection of WSMV.
文摘It has been reported that endosperm undergoes programmed cell death (PCD) during maize kernel development.Both bz1 (bronze ) and bz2 are anthocyanin biosynthetic genes,and related to development of aleuronic layer of maize seeds.Tyramide signal amplification fluorescence in situ hybridization (TSA FISH) is a novel and high sensitive FISH technique,which is suitable for routine application in plant cytogenetic research.Using this technique,we physically mapped the bz1 gene onto the short arm of chromosome 9 and the long arm of chromosome 1;the percentage distances from centromere to hybridization site were 40.2,75.4 respectively,and the bz2 onto the long arm of chromosome 1 and the short arm of chromosome 5;the percentage distances from centromere to hybridization site were 21.6,15.3 separately.The TSA FISH techniques of small low copy DNA sequences for plants are discussed.
