In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our...In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.展开更多
Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy ...Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy targets are not well understood.Methods:We conducted a systematic analysis using TCGA Pan-Cancer Atlas data and Gene Set Cancer Analysis results to examine the expression profiles of 15 DNA polymerases(POLYs)and their clinical correlations.We also evaluated the prognostic value of POLYs by analyzing their expression levels in relation to overall survival time(OS)using Kaplan-Meier survival curves.Additionally,we investigated the correlations between POLY expression and immune cells,DNA damage repair(DDR)pathways,and ubiquitination.Drug sensitivity analysis was performed to assess the relationship between POLY expression and drug response.Results:Our analysis revealed that 14 out of 15 POLYs exhibited significantly distinct expression patterns between tumor and normal samples across most cancer types,except for DNA nucleotidylexotransferase(DNTT).Specifically,POLD1 and POLE showed elevated expression in almost all cancers,while POLQ exhibited high expression levels in all cancer types.Some POLYs showed heightened expression in specific cancer subtypes,while others exhibited low expression.Kaplan-Meier survival curves demonstrated significant prognostic value of POLYs in multiple cancers,including PAAD,KIRC,and ACC.Cox analysis further validated these findings.Alteration patterns of POLYs varied significantly among different cancer types and were associated with poorer survival outcomes.Significant correlations were observed between the expression of POLY members and immune cells,DDR pathways,and ubiquitination.Drug sensitivity analysis indicated an inverse relationship between POLY expression and drug response.Conclusion:Our comprehensive study highlights the significant role of POLYs in cancer development and identifies them as promising prognostic and immunological biomarkers for various cancer types.Additionally,targeting POLYs therapeutically holds promise for tumor immunotherapy.展开更多
Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the sit...Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.展开更多
Nonsegmented negative-sense RNA viruses(nsNSVs)-including highly pathogenic pathogens such as measles virus(MeV),Nipah virus(NiV),Hendra virus(HeV),Ebola virus(EBOV),and others-pose major global health threats,yet mos...Nonsegmented negative-sense RNA viruses(nsNSVs)-including highly pathogenic pathogens such as measles virus(MeV),Nipah virus(NiV),Hendra virus(HeV),Ebola virus(EBOV),and others-pose major global health threats,yet most lack approved antiviral therapeutics.In the recent study,high-resolution cryo-electron microscopy(cryo-EM)revealed previously unrecognized allosteric pockets in the large(L)polymerase proteins of MeV and NiV,spatially distinct from the catalytic nucleotide-binding site.We further demonstrated that the non-nucleoside inhibitor ERDRP-0519 engages these pockets to allosterically‘lock’the polymerase in a mechanically inactive state.These findings reveal an allosteric mechanism of inhibition rooted in the conformational mechanics of the enzyme and highlight opportunities for integrating artificial intelligence(AI)-aided drug discovery(AIDD)into rational drug design.展开更多
Root organogenesis involves cell division, differentiation and expansion. The molecular mechanisms regulating root development are not fully understood. In this study, we identified poly（adenosine diphosphate （ADP）...Root organogenesis involves cell division, differentiation and expansion. The molecular mechanisms regulating root development are not fully understood. In this study, we identified poly（adenosine diphosphate （ADP）-ribose） polymerases （PARPs） as new players in root development. PARP catalyzes poly（ADP-ribosyl）ation of proteins by repeatedly adding ADP-ribose units onto proteins using nicotinamide adenine dinucleotide （NAD ） as the donor. We found that inhibition of PARP activities by 3-aminobenzomide （3-AB） increased the growth rates of both primary and lateral roots, leading to a more developed root system. The double mutant of Arabidopsis PARPs, parplparp2, showed more rapid primary and lateral root growth. Cyclin genes regulating G1-to-S and G2-to-M transition were up-regulated upon treatment by 3-AB. The proportion of 2C ceils increased while cells with higher DNA ploidy declined in the roots of treated plants, resulting in an enlarged root meristematic zone. The expression level of PARP2 was very low in the meristematic zone but high in the maturation zone, consistent with a role of PARP in inhibiting mitosis and promoting cell differentiation. Our results suggest that PARPs play an important role in root development by negatively regulating root cell division.展开更多
Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,inc...Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.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
Rapid diagnosis of Salmonella is crucial for the effective control of food safety incidents, especially in regions with poor hygiene conditions. Polymerase chain reaction(PCR), as a promising tool for Salmonella detec...Rapid diagnosis of Salmonella is crucial for the effective control of food safety incidents, especially in regions with poor hygiene conditions. Polymerase chain reaction(PCR), as a promising tool for Salmonella detection, is facing a lack of simple and fast sensing methods that are compatible with field applications in resource-limited areas. In this work, we developed a sensing approach to identify PCR-amplified Salmonella genomic DNA with the naked eye in a snapshot. Based on the ratiometric fiuorescence signals from SYBR Green Ⅰ and Hydroxyl naphthol blue, positive samples stood out from negative ones with a distinct color pattern under UV exposure. The proposed sensing scheme enabled highly specific identification of Salmonella with a detection limit at the single-copy level. Also, as a supplement to the intuitive naked-eye visualization results, numerical analysis of the colored images was available with a smartphone app to extract RGB values from colored images. This work provides a simple, rapid, and user-friendly solution for PCR identification, which promises great potential in molecular diagnosis of Salmonella and other pathogens in field.展开更多
Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are st...Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.展开更多
BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is...BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is the most frequently mutated site in HBV-related HCC;however,its prognostic significance is not fully established.AIM To evaluate the prognostic impact of TERT promoter mutations and efficiency of digital polymerase chain reaction(dPCR).METHODS A total of 66 HBV-related HCC patients who underwent hepatectomy were enrolled in this study.DNA extracted from fresh tumor tissues was analyzed for TERT promoter mutations using Sanger sequencing and dPCR.The dPCR assay was optimized by adding 7-deaza-dGTP,CviQ1,and ethylenediaminetetraacetic acid to improve detection sensitivity.Concordance between methods was assessed,and nomogram survival prediction models were developed to evaluate prognostic value based on mutation status.RESULTS TERT promoter mutations were detected in 26/66(39.39%)cases by Sanger sequencing and 30/66(45.45%)by dPCR.The two methods showed high concordance(93.939%,κ=0.876),with dPCR demonstrating 100%sensitivity and 90%specificity.Patients harboring TERT promoter mutations exhibited reduced overall survival and higher recurrence risk.Nomogram models successfully distinguished mutant from non-mutant cases for both overall survival(C-index:0.7651)and disease-free survival(C-index:0.6899).CONCLUSION TERT promoter mutation predicts poor prognosis in HBV-related HCC and serves as a biomarker for risk stratification.Optimized dPCR outperforms Sanger sequencing,and nomograms with TERT status guide precision therapy.展开更多
For the paper,Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus,published in Journal of Integrative Agriculture,2024 Vol.23,No.9,on pages 3145-3158,the authors'names and affiliat...For the paper,Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus,published in Journal of Integrative Agriculture,2024 Vol.23,No.9,on pages 3145-3158,the authors'names and affiliated units and Acknowledgements should be.展开更多
BACKGROUND Cytomegalovirus(CMV)is a ubiquitous herpesvirus that can cause significant ocular morbidity,particularly in immunocompromised individuals.AIM To summarize the current understanding of the ophthalmic impact ...BACKGROUND Cytomegalovirus(CMV)is a ubiquitous herpesvirus that can cause significant ocular morbidity,particularly in immunocompromised individuals.AIM To summarize the current understanding of the ophthalmic impact of CMV,with a focus on its epidemiology,clinical manifestations,diagnosis,and management,ocular symptoms of CMV floaters,blurred vision,and loss of peripheral vision,eventually progressing to retinal necrosis and detachment.CMV retinitis(CMVR)is a sight-threatening condition that can lead to retinal detachment,blindness,and even death.METHODS We discuss the pathophysiology of CMVR,including the role of immune suppression and viral reactivation.We also examine the clinical features of CMVR,including its characteristic retinal lesions and associated ocular complications.Diagnostic approaches are reviewed,including polymerase chain reaction and fundus photography.RESULTS We discuss treatment options,including antiviral medications,intravitreal injections,and surgical interventions.Finally,we highlight areas of ongoing research and future directions in managing CMV-related ocular disease.CONCLUSION CMV poses a significant threat to ocular health,particularly in immunocompromised populations such as those with human immunodeficiency virus/acquired immune deficiency syndrome.展开更多
We aim to study the semen carriage of human papillomavirus(HPV)and evaluate its association with patient characteristics.We conduct a single-center cohort study at Amiens University Hospital Center(Amiens,France).From...We aim to study the semen carriage of human papillomavirus(HPV)and evaluate its association with patient characteristics.We conduct a single-center cohort study at Amiens University Hospital Center(Amiens,France).From May 1 to October 31,2021,461 men consulting for infertility and with semen analysis data were included.Each participant gave his written informed consent for the use of laboratory,demographic,clinical,and lifestyle data.A proportion of the semen samples were sent to a virology laboratory for HPV screening in a polymerase chain reaction(PCR)assay.In univariate and multivariate analyses with a logistic regression model,HPV+and HPV−groups were compared with regard to semen characteristics(including the DNA fragmentation index and the sperm decondensation index)and demographic,clinical,and lifestyle variables.Semen HPV carriage was detected in 22.3% of the patients.High-oncogenic-risk HPV genotypes were predominant(57.6%).Multivariate analysis showed that HPV carriage was significantly associated with the presence of at least one abnormal spermogram dinging(according to the 6th World Health Organization criteria),with an adjusted odds ratio(OR)of 4.10(95%confidence interval[CI]:2.32-7.25,P<0.001).A statistically significant association was also found for the type of infertility(OR:1.61,95%CI:1.00-2.57,P=0.05),the presence of varicocele(OR:3.99,95%CI:1.48-10.71,P=0.01),and a history of cryptorchidism,testicular ectopia,or monorchidism(OR:3.54,95%CI:1.07-11.66,P=0.04).Infection with a single HPV genotype or multiple HPV genotypes was significantly associated with at least one abnormal spermogram finding for all HPV oncogenic risk groups(OR:3.93,95%CI:2.08-7.41,P<0.001;and OR:4.11,95%CI:1.58-10.68,P=0.01,respectively).The association between sperm HPV carriage and the risk of infertility was statistically significant in a multivariate analysis(OR:5.63,95%CI:3.16-10.01,P<0.001)and after adjustment for the propensity score(OR:6.10,95%CI:3.33-11.21,P<0.001).Our results suggest that semen HPV carriage has an impact on male fertility.Sperm screening for HPV might be a useful addition to the work-up for male infertility.展开更多
Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the...Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the established benchmarks for analyzing DNA mutations.However,the two methods necessitate intricate experimental preparation,costly instrumentation,and skilled personnel,making them challenging for rapid mutations analysis.More importantly,these methods lack adequate accuracy for one base mutations analysis[3].Therefore,the development of a reliable and exceptionally sensitive mutation analysis approach holds immense importance in cancer diagnosis and treatment.展开更多
The mitochondrial DNA copy number(mtDNAcn)plays a vital role in cellular energy metabolism and mitochondrial health.As mitochondria are responsible for adenosine triphosphate production through oxidative phosphorylati...The mitochondrial DNA copy number(mtDNAcn)plays a vital role in cellular energy metabolism and mitochondrial health.As mitochondria are responsible for adenosine triphosphate production through oxidative phosphorylation,maintaining an appropriate mtDNAcn level is vital for the overall cellular function.Alterations in mtDNAcn have been linked to various diseases,including neurodegenerative disorders,metabolic conditions,and cancers,making it an important biomarker for understanding the disease pathogenesis.The accurate estimation of mtDNAcn is essential for clinical applications.Quantitative polymerase chain reaction and next-generation sequencing are commonly employed techniques with distinct advantages and limitations.Clinically,mtDNAcn serves as a valuable indicator for early diagnosis,disease progression,and treatment response.For instance,in oncology,elevated mtDNAcn levels in blood samples are associated with tumor aggressiveness and can aid in monitoring treatment efficacy.In neurodegenerative diseases such as Alzheimer’s and Parkinson’s,altered mtDNAcn patterns provide insights into disease mechanisms and progression.Understanding and estimating mtDNAcn are critical for advancing diagnostic and therapeutic strategies in various medical fields.As research continues to uncover the implications of mtDNAcn alterations,its potential as a clinical biomarker is likely to expand,thereby enhancing our ability to diagnose and manage complex diseases.展开更多
Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease inclu...Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.展开更多
With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,ration...With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,rational optimization of the performance of DNA polymerase is of great interest.Nuclear magnetic resonance(NMR)spectroscopy is a powerful technique used for studying protein structure and dynamics.It provides the atomic resolution information of enzymes under their functional solution environment to reveal the active sites(hot spots)of the enzyme,which could be further used for optimizing the performance of enzymes.In our previous work,we identified hot spot residues of Pyrococcus furiosus DNA polymerase(Pfu pol).We aim to employ these binding hot spots to screen for co-factors of Pfu pol,particularly targeting those molecules exhibiting weak intermolecular interactions.To validate this concept,we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system.Employing these hot spots as probes,two new co-factors,the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine,are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol.This NMR-based approach requires no prior assignment information of target enzymes,guiding the rational exploration of novel cofactors for Pfu pol.Moreover,our approach is not dependent on structural data or bioinformatics.Therefore,it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.展开更多
TypeⅠinterferon(IFN)-mediated innate immune responses represent the first line of host defense against viral infection.However,the molecular mechanisms by which avian infuenza virus(AIV)inhibits typeⅠIFN production ...TypeⅠinterferon(IFN)-mediated innate immune responses represent the first line of host defense against viral infection.However,the molecular mechanisms by which avian infuenza virus(AIV)inhibits typeⅠIFN production in ducks are not well understood.Here,we frst found that the polymerase basic 2(PB2)protein of H5N1 subtype AIV inhibited the typeⅠIFN responses by targeting duck mitochondrial antiviral signaling protein(MAVS).We further demonstrated that H5N1-PB2 bound to theΔtransmembrane(ΔTM)domain of duck MAVS,and the polymerase basic 1(PB1)binding domain(PBD)and RNA binding nuclear import domain(RND)of H5N1-PB2 interacted with MAVS to inhibit typeⅠIFN expression in ducks.Collectively,our fndings contribute to understanding the molecular mechanism by which AIV proteins regulate the retinoic acid-inducible geneⅠ(RIG-Ⅰ)-like receptor(RLR)signaling pathway to evade host antiviral immune responses in ducks.展开更多
BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To ...BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To identify POLE non-EDMs and EDMs in CRC,and to determine their associations with accompanying mutations and microsatellite instability(MSI).METHODS In this retrospective study,next-generation sequencing was performed using a targeted colon cancer panel(Qiagen,DHS-003Z)on 356 CRC patients.Of these,191 patients were found to carry POLE mutations.For these patients,MSI status was assessed using both real-time PCR(EasyPGX^(■)Ready MSI kit)and immunohistochemistry,and accompanying somatic mutations were investigated.RESULTS POLE mutations were identified in 53.65%of the CRC patients.Among the POLE-mutant patients,87.96%were classified as pMMR(MSI-L),and 12.04%as dMMR(MSI-H).The most frequently observed POLE non-EDM variant was exon 34 c.4337_4338delTG p.V1446fs*3.The POLE EDMs were present in exon 14,with two specific variants p.Y458F(0.52%)and p.Y468N(0.52%).The most common pathogenic variants accompanying the POLE mutations were in MLH3,MSH3,KRAS,PIK3CA,and BRAF genes.POLE mutations were associated with a high mutational burden and MSI in CRC,particularly in the dMMR phenotype.This association suggests that POLE mutations may serve as important biomarkers for understanding the genetic profile of the disease and may be used in the clinical management of CRC.CONCLUSION POLE mutations,especially non-EDMs,are frequent in MSI-L CRC and often co-occur with MLH3,MSH3,KRAS,PIK3CA,and BRAF,highlighting their potential role in tumor biology and as biomarkers for personalized treatment.Functional validation and multicenter studies are needed.展开更多
BACKGROUND Necrotizing enterocolitis(NEC)remains a prominent gastrointestinal emergency among infants,particularly term infants with congenital heart defects(CHD)being at high risk.The molecular processes that contrib...BACKGROUND Necrotizing enterocolitis(NEC)remains a prominent gastrointestinal emergency among infants,particularly term infants with congenital heart defects(CHD)being at high risk.The molecular processes that contribute to NEC have yet to be completely understood.The high mortality rates necessitate an active search for noninvasive biomarkers that can aid in the preclinical diagnosis and prognosis of NEC.MicroRNAs(miRs),which are involved in many biological processes in both health and disease,have been discovered to play an important role in regulating inflammation and immune responses via various signaling pathways.AIM To determine the plasma levels of miR-155,miR-221,miR-223,miR-320a,miR-451a as potential NEC biomarkers in term newborns with CHD.METHODS This prospective cohort study included twenty-tree term newborns with CHD who underwent cardiac surgery on the median day of life(DOL)=7.Nine of them developed NEC(Bell’s stage IIA and IIIA)within 1 week of cardiac surgery(NEC newborns).Blood samples were collected before(median DOL=5)and following(median DOL=13)cardiac surgery.Levels of plasma miR-155-5p,miR-221-3p,miR-223-3p,miR-320a-3p,and miR-451a were determined using real-time polymerase chain reaction.The functional analysis was executed using the DIANA-miRPath v4.0.RESULTS Preoperatively,NEC newborns had significantly lower plasma levels of miR-155(2.70-fold,P=0.020),miR-223(2.42-fold,P=0.030),and miR-320a(3.62-fold,P=0.006)than newborns without NEC.Postoperatively,miR-451a levels differed significantly between the newborn groups,showing a 4.70-fold decrease(P=0.014)in expression when clinical NEC symptoms appeared.According to receiver operating characteristic analysis,miR-320a was found to be the most effective predictive biomarker for NEC[area under the curve(AUC)=0.835,63%sensitivity,100%specificity],while miR-451a was identified as a NEC biomarker(AUC=0.835,85.7%sensitivity,76.9%specificity).Preoperatively,miR-155-5p,miR-223-3p,and miR-320a-3p were differentially expressed and targeted the forkhead box O and Hippo pathways(P<0.01).CONCLUSION Our study demonstrates,for the first time,that plasma miR-320a-3p levels can be used as a preclinical biomarker for NEC in term newborns with CHD.展开更多
文摘In their seminal publication describing the structure of the DNA double helix , Watson and Crick wrote what may be one of the greatest understatements in the scientific literature, namely that ＂It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.＂ Half a century later, we more fully appreciate what a huge challenge it is to replicate six billion nucleotides with the accuracy needed to stably maintain the human genome over many generations. This challenge is perhaps greater than was realized 50 years ago, because subsequent studies have revealed that the genome can be destabilized not only by environmental stresses that generate a large number and variety of potentially cytotoxic and mutagenic lesions in DNA but also by various sequence motifs of normal DNA that present challenges to replication. Towards a better understanding of the many determinants of genome stability, this chapter reviews the fidelity with which undamaged and damaged DNA is copied, with a focus on the eukaryotic B- and Y-family DNA polymerases, and considers how this fidelity is achieved.
基金supported by the project of funds by the Consultation of Provincial Department and University for S&T Innovation granted by Hebei Provincial Department of Science and Technology and Hebei Medical University(2020TXZH04).
文摘Introduction:DNA polymerases are crucial for maintaining genome stability and influencing tumorigenesis.However,the clinical implications of DNA polymerases in tumorigenesis and their potential as anti-cancer therapy targets are not well understood.Methods:We conducted a systematic analysis using TCGA Pan-Cancer Atlas data and Gene Set Cancer Analysis results to examine the expression profiles of 15 DNA polymerases(POLYs)and their clinical correlations.We also evaluated the prognostic value of POLYs by analyzing their expression levels in relation to overall survival time(OS)using Kaplan-Meier survival curves.Additionally,we investigated the correlations between POLY expression and immune cells,DNA damage repair(DDR)pathways,and ubiquitination.Drug sensitivity analysis was performed to assess the relationship between POLY expression and drug response.Results:Our analysis revealed that 14 out of 15 POLYs exhibited significantly distinct expression patterns between tumor and normal samples across most cancer types,except for DNA nucleotidylexotransferase(DNTT).Specifically,POLD1 and POLE showed elevated expression in almost all cancers,while POLQ exhibited high expression levels in all cancer types.Some POLYs showed heightened expression in specific cancer subtypes,while others exhibited low expression.Kaplan-Meier survival curves demonstrated significant prognostic value of POLYs in multiple cancers,including PAAD,KIRC,and ACC.Cox analysis further validated these findings.Alteration patterns of POLYs varied significantly among different cancer types and were associated with poorer survival outcomes.Significant correlations were observed between the expression of POLY members and immune cells,DDR pathways,and ubiquitination.Drug sensitivity analysis indicated an inverse relationship between POLY expression and drug response.Conclusion:Our comprehensive study highlights the significant role of POLYs in cancer development and identifies them as promising prognostic and immunological biomarkers for various cancer types.Additionally,targeting POLYs therapeutically holds promise for tumor immunotherapy.
文摘Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.
基金supported by a grant from ShanghaiTech University to H.Z.,and a grant from the Shanxi Key Laboratory of Protein Structure Determination(202104010910006)to H.Z.
文摘Nonsegmented negative-sense RNA viruses(nsNSVs)-including highly pathogenic pathogens such as measles virus(MeV),Nipah virus(NiV),Hendra virus(HeV),Ebola virus(EBOV),and others-pose major global health threats,yet most lack approved antiviral therapeutics.In the recent study,high-resolution cryo-electron microscopy(cryo-EM)revealed previously unrecognized allosteric pockets in the large(L)polymerase proteins of MeV and NiV,spatially distinct from the catalytic nucleotide-binding site.We further demonstrated that the non-nucleoside inhibitor ERDRP-0519 engages these pockets to allosterically‘lock’the polymerase in a mechanically inactive state.These findings reveal an allosteric mechanism of inhibition rooted in the conformational mechanics of the enzyme and highlight opportunities for integrating artificial intelligence(AI)-aided drug discovery(AIDD)into rational drug design.
基金supported by grants to X.G. from the National Natural Science Foundation of China(31170169 and 31070232)
文摘Root organogenesis involves cell division, differentiation and expansion. The molecular mechanisms regulating root development are not fully understood. In this study, we identified poly（adenosine diphosphate （ADP）-ribose） polymerases （PARPs） as new players in root development. PARP catalyzes poly（ADP-ribosyl）ation of proteins by repeatedly adding ADP-ribose units onto proteins using nicotinamide adenine dinucleotide （NAD ） as the donor. We found that inhibition of PARP activities by 3-aminobenzomide （3-AB） increased the growth rates of both primary and lateral roots, leading to a more developed root system. The double mutant of Arabidopsis PARPs, parplparp2, showed more rapid primary and lateral root growth. Cyclin genes regulating G1-to-S and G2-to-M transition were up-regulated upon treatment by 3-AB. The proportion of 2C ceils increased while cells with higher DNA ploidy declined in the roots of treated plants, resulting in an enlarged root meristematic zone. The expression level of PARP2 was very low in the meristematic zone but high in the maturation zone, consistent with a role of PARP in inhibiting mitosis and promoting cell differentiation. Our results suggest that PARPs play an important role in root development by negatively regulating root cell division.
基金funded by the grants from the National Key R&D Program of China(2021YFC2300200 to Q.P.and 2021YFC2300700 to Y.S.)Strategic Priority Research Program of CAS(XDB29010000 to Y.S.)+1 种基金National Natural Science Foundation of China(NSFC)(81871658 and 32192452 to Y.S.and 32100119 to Q.P.)Y.S.is also partially supported by the Youth Innovation Promotion Association of CAS(Y201921).
文摘Jingmenviruses are a group of flavi-like viruses with segmented genome and have been found in various types of hosts,including humans,cattle,monkeys,bats,rodents,sheep,ticks,mosquitoes and nematodes.Jingmenviruses,including the Jingmen tick virus(JMTV)and Alongshan virus(ALSV),have been associated with febrile illness and flu-like symptoms in humans.Viral polymerase plays critical roles in genome replication and transcription and is an ideal target for antiviral drugs.Here,we determined the crystal structures of RNA-dependent RNA polymerase(RdRp)domains of JMTV and ALSV at 2.6Åand 3.2Åresolutions,respectively.The overall structures of JMTV and ALSV RdRp domains are similar to those from the typical unsegmented viruses in Flaviviridae family,especially the Flavivirus genus.JMTV and ALSV RdRps can be divided into three subdomains and the catalytical Motif A-G are conserved like the typical flaviviruses,whereas the zinc-binding pockets are absent from the JMTV and ALSV RdRps.The 50-ends of jingmenvirus genomes are varied in length and sequence,and a highly conserved 8-nucleotide element located on the tip of stem loop A was identified and shown to be required for binding with RdRp and performing de novo replication activity.These findings provide important structural insights into RdRp of segmented flavivirus and reveal the key region of virus genome responsible for replication initiation,which would promote molecular understanding of segmented flavivirus replication and the structure-based design of antiviral drugs against flaviviruses.
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 Macao Science and Technology Development Fund(FDCT)(Nos.FDCT 0029/2021/A1,FDCT0002/2021/AKP,004/2023/SKL,0036/2021/APD)University of Macao(No.MYRG-GRG2023-00034-IME,SRG2024-00057IME)+2 种基金Dr.Stanley Ho Medical Development Foundation(No.SHMDF-OIRFS/2024/001)Zhuhai Huafa Group(No.HF-006-2021)Guangdong Science and Technology Department(No.2022A0505030022)。
文摘Rapid diagnosis of Salmonella is crucial for the effective control of food safety incidents, especially in regions with poor hygiene conditions. Polymerase chain reaction(PCR), as a promising tool for Salmonella detection, is facing a lack of simple and fast sensing methods that are compatible with field applications in resource-limited areas. In this work, we developed a sensing approach to identify PCR-amplified Salmonella genomic DNA with the naked eye in a snapshot. Based on the ratiometric fiuorescence signals from SYBR Green Ⅰ and Hydroxyl naphthol blue, positive samples stood out from negative ones with a distinct color pattern under UV exposure. The proposed sensing scheme enabled highly specific identification of Salmonella with a detection limit at the single-copy level. Also, as a supplement to the intuitive naked-eye visualization results, numerical analysis of the colored images was available with a smartphone app to extract RGB values from colored images. This work provides a simple, rapid, and user-friendly solution for PCR identification, which promises great potential in molecular diagnosis of Salmonella and other pathogens in field.
基金the Experimental Technology Research Project of Zhejiang University(SYB202138)National Natural Science Foundation of China(32000195).
文摘Soil DNA extraction,such as microbial community analysis and gene drift detection,is an important basis for multiple analyses in different fields.Nevertheless,the soil DNA extraction methods for field detection are still lacking.This study established a rapid soil DNA extraction(RSDE)method that can be used in field detection.In this method,we first utilized the optimized lysate to isolate DNA from soil and then used a filtration membrane and a DNA adsorption membrane to purify the DNA via the column method.Moreover,we used the pressure from the syringe instead of the conventional centrifugal force of the centrifuge to assist the sample filtration,resulting in very low requirements for this method,with an extraction time of less than 20 min.Furthermore,we demonstrated that the RSDE method was applicable for DNA extraction from different types of soils,with the demand for soil samples as low as 0.1 g and that the amount of obtained DNA was,to some extent,greater than that obtained by a commercial kit.Further analysis revealed that this extracted genomic DNA can be used directly for polymerase chain reaction(PCR)analysis,including ordinary PCR,real-time fluorescent quantitative PCR,and recombinase polymerase amplification(RPA)-CRISPR/Cas12a visual assays.In addition,we demonstrated that this method can be used to extract DNA from residual plant roots in addition to soil microbes,which lays a foundation for the comprehensive analysis of soil plants and microorganisms.In summary,the RSDE method proposed in this study may have wide application prospects.
基金Supported by National Key Research and Development Program of China,No.2023YFF0613304Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences,No.2023-I2M-2-004,No.2024-I2M-C&T-B-069,and No.2025-I2M-C&T-B-057.
文摘BACKGROUND Recurrence remains the leading cause of poor prognosis in hepatocellular carcinoma(HCC),particularly among patients infected with hepatitis B virus(HBV).The telomerase reverse transcriptase(TERT)promoter is the most frequently mutated site in HBV-related HCC;however,its prognostic significance is not fully established.AIM To evaluate the prognostic impact of TERT promoter mutations and efficiency of digital polymerase chain reaction(dPCR).METHODS A total of 66 HBV-related HCC patients who underwent hepatectomy were enrolled in this study.DNA extracted from fresh tumor tissues was analyzed for TERT promoter mutations using Sanger sequencing and dPCR.The dPCR assay was optimized by adding 7-deaza-dGTP,CviQ1,and ethylenediaminetetraacetic acid to improve detection sensitivity.Concordance between methods was assessed,and nomogram survival prediction models were developed to evaluate prognostic value based on mutation status.RESULTS TERT promoter mutations were detected in 26/66(39.39%)cases by Sanger sequencing and 30/66(45.45%)by dPCR.The two methods showed high concordance(93.939%,κ=0.876),with dPCR demonstrating 100%sensitivity and 90%specificity.Patients harboring TERT promoter mutations exhibited reduced overall survival and higher recurrence risk.Nomogram models successfully distinguished mutant from non-mutant cases for both overall survival(C-index:0.7651)and disease-free survival(C-index:0.6899).CONCLUSION TERT promoter mutation predicts poor prognosis in HBV-related HCC and serves as a biomarker for risk stratification.Optimized dPCR outperforms Sanger sequencing,and nomograms with TERT status guide precision therapy.
文摘For the paper,Role of feline ANP32 proteins in regulating polymerase activity of influenza A virus,published in Journal of Integrative Agriculture,2024 Vol.23,No.9,on pages 3145-3158,the authors'names and affiliated units and Acknowledgements should be.
文摘BACKGROUND Cytomegalovirus(CMV)is a ubiquitous herpesvirus that can cause significant ocular morbidity,particularly in immunocompromised individuals.AIM To summarize the current understanding of the ophthalmic impact of CMV,with a focus on its epidemiology,clinical manifestations,diagnosis,and management,ocular symptoms of CMV floaters,blurred vision,and loss of peripheral vision,eventually progressing to retinal necrosis and detachment.CMV retinitis(CMVR)is a sight-threatening condition that can lead to retinal detachment,blindness,and even death.METHODS We discuss the pathophysiology of CMVR,including the role of immune suppression and viral reactivation.We also examine the clinical features of CMVR,including its characteristic retinal lesions and associated ocular complications.Diagnostic approaches are reviewed,including polymerase chain reaction and fundus photography.RESULTS We discuss treatment options,including antiviral medications,intravitreal injections,and surgical interventions.Finally,we highlight areas of ongoing research and future directions in managing CMV-related ocular disease.CONCLUSION CMV poses a significant threat to ocular health,particularly in immunocompromised populations such as those with human immunodeficiency virus/acquired immune deficiency syndrome.
文摘We aim to study the semen carriage of human papillomavirus(HPV)and evaluate its association with patient characteristics.We conduct a single-center cohort study at Amiens University Hospital Center(Amiens,France).From May 1 to October 31,2021,461 men consulting for infertility and with semen analysis data were included.Each participant gave his written informed consent for the use of laboratory,demographic,clinical,and lifestyle data.A proportion of the semen samples were sent to a virology laboratory for HPV screening in a polymerase chain reaction(PCR)assay.In univariate and multivariate analyses with a logistic regression model,HPV+and HPV−groups were compared with regard to semen characteristics(including the DNA fragmentation index and the sperm decondensation index)and demographic,clinical,and lifestyle variables.Semen HPV carriage was detected in 22.3% of the patients.High-oncogenic-risk HPV genotypes were predominant(57.6%).Multivariate analysis showed that HPV carriage was significantly associated with the presence of at least one abnormal spermogram dinging(according to the 6th World Health Organization criteria),with an adjusted odds ratio(OR)of 4.10(95%confidence interval[CI]:2.32-7.25,P<0.001).A statistically significant association was also found for the type of infertility(OR:1.61,95%CI:1.00-2.57,P=0.05),the presence of varicocele(OR:3.99,95%CI:1.48-10.71,P=0.01),and a history of cryptorchidism,testicular ectopia,or monorchidism(OR:3.54,95%CI:1.07-11.66,P=0.04).Infection with a single HPV genotype or multiple HPV genotypes was significantly associated with at least one abnormal spermogram finding for all HPV oncogenic risk groups(OR:3.93,95%CI:2.08-7.41,P<0.001;and OR:4.11,95%CI:1.58-10.68,P=0.01,respectively).The association between sperm HPV carriage and the risk of infertility was statistically significant in a multivariate analysis(OR:5.63,95%CI:3.16-10.01,P<0.001)and after adjustment for the propensity score(OR:6.10,95%CI:3.33-11.21,P<0.001).Our results suggest that semen HPV carriage has an impact on male fertility.Sperm screening for HPV might be a useful addition to the work-up for male infertility.
文摘Dear Editor,Mutations in genomic sequences exhibit a strong correlation with various pathological processes of cancers[1].Currently,the next-generation sequencing technique[2]and polymerase chain reaction(PCR)were the established benchmarks for analyzing DNA mutations.However,the two methods necessitate intricate experimental preparation,costly instrumentation,and skilled personnel,making them challenging for rapid mutations analysis.More importantly,these methods lack adequate accuracy for one base mutations analysis[3].Therefore,the development of a reliable and exceptionally sensitive mutation analysis approach holds immense importance in cancer diagnosis and treatment.
文摘The mitochondrial DNA copy number(mtDNAcn)plays a vital role in cellular energy metabolism and mitochondrial health.As mitochondria are responsible for adenosine triphosphate production through oxidative phosphorylation,maintaining an appropriate mtDNAcn level is vital for the overall cellular function.Alterations in mtDNAcn have been linked to various diseases,including neurodegenerative disorders,metabolic conditions,and cancers,making it an important biomarker for understanding the disease pathogenesis.The accurate estimation of mtDNAcn is essential for clinical applications.Quantitative polymerase chain reaction and next-generation sequencing are commonly employed techniques with distinct advantages and limitations.Clinically,mtDNAcn serves as a valuable indicator for early diagnosis,disease progression,and treatment response.For instance,in oncology,elevated mtDNAcn levels in blood samples are associated with tumor aggressiveness and can aid in monitoring treatment efficacy.In neurodegenerative diseases such as Alzheimer’s and Parkinson’s,altered mtDNAcn patterns provide insights into disease mechanisms and progression.Understanding and estimating mtDNAcn are critical for advancing diagnostic and therapeutic strategies in various medical fields.As research continues to uncover the implications of mtDNAcn alterations,its potential as a clinical biomarker is likely to expand,thereby enhancing our ability to diagnose and manage complex diseases.
基金Supported by the Visiting and Training Foundation of Teachers in Ordinary Undergraduate Universities of Shandong Province,the Qingdao Agricultural University Doctoral Start-Up Fund(No.6631122030)the Advanced Talents Foundation of QAU(No.6651118016)+2 种基金the Fish Innovation Team of Shandong Agriculture Research System(No.SDAIT12-06)the Shandong Engineering Research Center for Prevention and Control of Aquatic Animal Disease,the“First Class Fishery Discipline”Program[(2020)3]of Shandong Provincethe Key R&D Program(Soft Science Project)of Shandong Province,China(No.2023 RKY 06004)。
文摘Glugea plecoglossi,a microsporidia of the Glugea genus,can cause an infamous disease Plecoglossus altivelis in East Asia,resulting in heavy economic losses.At present,the main diagnostic methods for this disease include microscopy examination,quantitative real-time PCR,and loop-mediated isothermal amplification-lateral flow dipstick(LAMP-LFD).In this study,a recombinase polymerase amplification-lateral flow dipstick(RPA-LFD)method,targeting the beta-tubulin gene,was developed to detect G.plecoglossi,three sets of primers and probes were designed and screened,after which the initial reaction system was established.The RPA-LFD method for G.plecoglossi could complete nucleic acid amplification at 39℃ for 10 min,after which the amplification product was dropped on the LFD strip,and the results could then be observed within 5 min.A specificity assay revealed that there was no cross reactivity with other protozoa except G.plecoglossi.A sensitivity assay revealed that the detection limit was 9.38×10^(-6) ng/μL,which was more sensitive than that of conventional PCR.Compared with conventional detection methods,the novel RPA-LFD method has the advantages of simple operation,short operation time,high sensitivity,and high specificity for G.plecoglossi detection,indicating its potential use in rapid field detection of G.plecoglossi.
基金supported by the Strategic Priority Research Program of the Chinese Academy of Sciences XDB0540000Natural Science Foundation of China grants 22327901,22174151 and 21991080Hubei Provincial Natural Science Foundation of China 2023AFA041。
文摘With rapid developments of emerging technologies like synthetic biology,the demand for DNA polymerases with superior activities including higher thermostability and processivity has increased significantly.Thus,rational optimization of the performance of DNA polymerase is of great interest.Nuclear magnetic resonance(NMR)spectroscopy is a powerful technique used for studying protein structure and dynamics.It provides the atomic resolution information of enzymes under their functional solution environment to reveal the active sites(hot spots)of the enzyme,which could be further used for optimizing the performance of enzymes.In our previous work,we identified hot spot residues of Pyrococcus furiosus DNA polymerase(Pfu pol).We aim to employ these binding hot spots to screen for co-factors of Pfu pol,particularly targeting those molecules exhibiting weak intermolecular interactions.To validate this concept,we first demonstrated the feasibility of utilizing hot spot residues as screening probes for auxiliary factors by employing the well-characterized Tween-20 as a model system.Employing these hot spots as probes,two new co-factors,the heat shock protein TkHSP20 from Thermococcus Kodakaraensis and the chemical chaperone L-arginine,are identified to interact with Pfu pol to boost its performance in amplifying long DNA fragments by enhancing the thermal stability and the processivity of the Pfu pol.This NMR-based approach requires no prior assignment information of target enzymes,guiding the rational exploration of novel cofactors for Pfu pol.Moreover,our approach is not dependent on structural data or bioinformatics.Therefore,it has significant potential for application in various enzymes to expedite the progress in enzyme engineering.
基金supported by the grants from the National Natural Science Foundation of China(31872497 and 32072844)the National Key Research and Development Program of China(2021YFD1800200 and 2016YFD0500207)the Laboratory of Lingnan Modern Agriculture Project,China(NT2021007)。
文摘TypeⅠinterferon(IFN)-mediated innate immune responses represent the first line of host defense against viral infection.However,the molecular mechanisms by which avian infuenza virus(AIV)inhibits typeⅠIFN production in ducks are not well understood.Here,we frst found that the polymerase basic 2(PB2)protein of H5N1 subtype AIV inhibited the typeⅠIFN responses by targeting duck mitochondrial antiviral signaling protein(MAVS).We further demonstrated that H5N1-PB2 bound to theΔtransmembrane(ΔTM)domain of duck MAVS,and the polymerase basic 1(PB1)binding domain(PBD)and RNA binding nuclear import domain(RND)of H5N1-PB2 interacted with MAVS to inhibit typeⅠIFN expression in ducks.Collectively,our fndings contribute to understanding the molecular mechanism by which AIV proteins regulate the retinoic acid-inducible geneⅠ(RIG-Ⅰ)-like receptor(RLR)signaling pathway to evade host antiviral immune responses in ducks.
文摘BACKGROUND Although the relationship between somatic DNA polymerase epsilon(POLE)exonuclease domain mutations(EDMs)and colorectal cancer(CRC)is well established,the role of POLE non-EDMs in CRC remains unclear.AIM To identify POLE non-EDMs and EDMs in CRC,and to determine their associations with accompanying mutations and microsatellite instability(MSI).METHODS In this retrospective study,next-generation sequencing was performed using a targeted colon cancer panel(Qiagen,DHS-003Z)on 356 CRC patients.Of these,191 patients were found to carry POLE mutations.For these patients,MSI status was assessed using both real-time PCR(EasyPGX^(■)Ready MSI kit)and immunohistochemistry,and accompanying somatic mutations were investigated.RESULTS POLE mutations were identified in 53.65%of the CRC patients.Among the POLE-mutant patients,87.96%were classified as pMMR(MSI-L),and 12.04%as dMMR(MSI-H).The most frequently observed POLE non-EDM variant was exon 34 c.4337_4338delTG p.V1446fs*3.The POLE EDMs were present in exon 14,with two specific variants p.Y458F(0.52%)and p.Y468N(0.52%).The most common pathogenic variants accompanying the POLE mutations were in MLH3,MSH3,KRAS,PIK3CA,and BRAF genes.POLE mutations were associated with a high mutational burden and MSI in CRC,particularly in the dMMR phenotype.This association suggests that POLE mutations may serve as important biomarkers for understanding the genetic profile of the disease and may be used in the clinical management of CRC.CONCLUSION POLE mutations,especially non-EDMs,are frequent in MSI-L CRC and often co-occur with MLH3,MSH3,KRAS,PIK3CA,and BRAF,highlighting their potential role in tumor biology and as biomarkers for personalized treatment.Functional validation and multicenter studies are needed.
基金Supported by The Russian Science Foundation,No.19-75-20076.
文摘BACKGROUND Necrotizing enterocolitis(NEC)remains a prominent gastrointestinal emergency among infants,particularly term infants with congenital heart defects(CHD)being at high risk.The molecular processes that contribute to NEC have yet to be completely understood.The high mortality rates necessitate an active search for noninvasive biomarkers that can aid in the preclinical diagnosis and prognosis of NEC.MicroRNAs(miRs),which are involved in many biological processes in both health and disease,have been discovered to play an important role in regulating inflammation and immune responses via various signaling pathways.AIM To determine the plasma levels of miR-155,miR-221,miR-223,miR-320a,miR-451a as potential NEC biomarkers in term newborns with CHD.METHODS This prospective cohort study included twenty-tree term newborns with CHD who underwent cardiac surgery on the median day of life(DOL)=7.Nine of them developed NEC(Bell’s stage IIA and IIIA)within 1 week of cardiac surgery(NEC newborns).Blood samples were collected before(median DOL=5)and following(median DOL=13)cardiac surgery.Levels of plasma miR-155-5p,miR-221-3p,miR-223-3p,miR-320a-3p,and miR-451a were determined using real-time polymerase chain reaction.The functional analysis was executed using the DIANA-miRPath v4.0.RESULTS Preoperatively,NEC newborns had significantly lower plasma levels of miR-155(2.70-fold,P=0.020),miR-223(2.42-fold,P=0.030),and miR-320a(3.62-fold,P=0.006)than newborns without NEC.Postoperatively,miR-451a levels differed significantly between the newborn groups,showing a 4.70-fold decrease(P=0.014)in expression when clinical NEC symptoms appeared.According to receiver operating characteristic analysis,miR-320a was found to be the most effective predictive biomarker for NEC[area under the curve(AUC)=0.835,63%sensitivity,100%specificity],while miR-451a was identified as a NEC biomarker(AUC=0.835,85.7%sensitivity,76.9%specificity).Preoperatively,miR-155-5p,miR-223-3p,and miR-320a-3p were differentially expressed and targeted the forkhead box O and Hippo pathways(P<0.01).CONCLUSION Our study demonstrates,for the first time,that plasma miR-320a-3p levels can be used as a preclinical biomarker for NEC in term newborns with CHD.
