鹅星状病毒RdRp蛋白对细胞因子转录水平的影响

为了解鹅星状病毒(GAstV)RdRp蛋白对细胞因子转录水平的影响,试验将构建的pCMV-RdRp真核质粒转染293细胞,并于转染后12、24、36 h收集细胞,提取核酸检测细胞内CCL2、CCL5、IL-1β、IFN-α、TNF-α、IFITM1和IFITM2的转录水平。结果发现,GAstV RdRp蛋白于12、24、36 h极显著抑制CCL2转录,12 h极显著促进CCL5表达,24、36 h极显著抑制CCL5表达(P<0.01);GAstV RdRp蛋白于24、36 h极显著促进IL-1β转录(P<0.01);GAstV RdRp蛋白于12 h极显著促进IFN-α的转录,于24、36 h极显著抑制IFN-α转录(P<0.01);GAstV RdRp蛋白于12、24、36 h显著或极显著抑制TNF-α转录(P<0.01);GAstV RdRp蛋白于24 h极显著抑制IFITM1转录(P<0.01),于36 h显著抑制IFITM1转录(P<0.05),于12、36 h极显著促进IFITM2转录(P<0.01),于24 h极显著抑制IFITM2的转录(P<0.01)。表明GAstV RdRp蛋白过表达能够诱导IL-1β的转录,抑制CCL2、TNF-α和IFITM1的转录;GAstV RdRp蛋白早期促进CCL5和IFN-α的转录,后期抑制CCL5和IFN-α的转录;GAstV RdRp蛋白早期和晚期促进IFITM2转录,中期抑制IFITM2转录。

Systematic analysis of DNA polymerases as therapeutic targets in pan-cancers

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.

Development of a multiplex polymerase chain reaction assay for detection of hepatitis C virus,hepatitis B virus,and human immunodeficiency virus 1

BACKGROUND Hepatitis C virus(HCV),hepatitis B virus(HBV),and human immunodeficiency virus 1(HIV-1)are the most epidemic blood-borne viruses,posing threats to human health and causing economic losses to nations for combating the infection transmission.The diagnostic methodologies that depend on the detection of viral nucleic acids are much more expensive,but they are more accurate than sero-logical testing.AIM To develop a rapid,cost-effective,and accurate diagnostic multiplex polymerase chain reaction(PCR)assay for simultaneous detection of HCV,HBV,and HIV-1.METHODS The design of the proposed PCR assay targets the amplification of a short conserved region featured with a distinguishable melting profile and electro-phoretic molecular weight inside each viral genome.Therefore,this diagnostic method will be appropriate for application in both conventional(combined with electrophoresis)and real-time PCR facilities.Confirmatory in silico investigations were conducted to prove the capability of the approached PCR assay to detect variants of each virus.Then,Egyptian isolates of each virus were subjected to the wet lab examination using the given diagnostic assay.RESULTS The in silico investigations confirmed that the PCR primers can match many viral variants in a multiplex PCR assay.The wet lab experiment proved the efficiency of the assay in distinguishing each viral type through high-resolution melting analysis.Compared to related published assays,the proposed assay in the current study is more sensitive and competitive with many expensive PCR assays.CONCLUSION This study provides a simple,cost-effective,and sensitive diagnostic PCR assay facilitating the detection of the most epidemic blood-borne viruses;this makes the proposed assay promising to be substitutive for the mistakable and cheap serological-based assays.

Low testing rates and high BRCA prevalence: Poly (ADP-ribose) polymerase inhibitor use in Middle East BRCA/homologous recombination deficiency-positive cancer patients

BACKGROUND Poly(ADP-ribose)polymerase inhibitors(PARPis)are approved as first-line therapies for breast cancer gene(BRCA)-positive,human epidermal growth factor receptor 2-negative locally advanced or metastatic breast cancer.They are also effective for new and recurrent ovarian cancers that are BRCA-or homologous recombination deficiency(HRD)-positive.However,data on these mutations and PARPi use in the Middle East are limited.AIM To assess BRCA/HRD prevalence and PARPi use in patients in the Middle East with breast/ovarian cancer.METHODS This was a single-center retrospective study of 57 of 472 breast cancer patients tested for BRCA mutations,and 25 of 65 ovarian cancer patients tested for HRD.These adult patients participated in at least four visits to the oncology service at our center between August 2021 and May 2023.Data were summarized using descriptive statistics and compared using counts and percentages.Response to treatment was assessed using Response Evaluation Criteria in Solid Tumors criteria.RESULTS Among the 472 breast cancer patients,12.1%underwent BRCA testing,and 38.5%of 65 ovarian cancer patients received HRD testing.Pathogenic mutations were found in 25.6%of the tested patients:26.3%breast cancers had germline BRCA(gBRCA)mutations and 24.0%ovarian cancers showed HRD.Notably,40.0%of gBRCA-positive breast cancers and 66.0%of HRD-positive ovarian cancers were Middle Eastern and Asian patients,respectively.PARPi treatment was used in 5(33.3%)gBRCA-positive breast cancer patients as first-line therapy(n=1;7-months progression-free),for maintenance(n=2;>15-months progression-free),or at later stages due to compliance issues(n=2).Four patients(66.6%)with HRD-positive ovarian cancer received PARPi and all remained progression-free.CONCLUSION Lower testing rates but higher BRCA mutations in breast cancer were found.Ethnicity reflected United Arab Emirates demographics,with breast cancer in Middle Eastern and ovarian cancer in Asian patients.

两株GI.1型和GI.2型兔出血症病毒RdRp基因的克隆与分析

为了解兔出血症病毒(rabbit hemorrhagic disease virus,RHDV)RdRp基因的遗传稳定性和变异规律,本研究分别克隆了RHDV SCH04株和RHDV2 SCCN03株的RdRp基因,然后对基因序列进行比对和系统发育分析,并通过编码蛋白质的理化性质、信号肽、跨膜区域、二级结构、三级结构、磷酸化和糖基化修饰位点的预测,对RdRp进行生物信息学分析。结果显示,两株RdRp基因序列全长均为1548 bp,编码516个氨基酸。GI.1型RHDV毒株间序列相似性为86.9%~99.9%,GI.2型RHDV毒株间为91.3%~99.9%。GI.1型和GI.2型RHDV毒株间序列相似性为84.3%~88.3%。SCH04株和SCCN03株分别属于GI.1a型和GI.2型RHDV,且SCH04株和SCCN03株的RdRp基因核苷酸序列相似性为84.95%,其编码蛋白存在17个氨基酸差异位点,氨基酸序列相似性为96.71%;二者均属于稳定蛋白,不存在信号肽和跨膜区域;其分子量、等电点、脂溶系数、平均亲水系数和不稳定系数等指标无显著差异。SCH04株RdRp的α螺旋和β转角比例比SCCN03株略高,分别为41.86%和4.26%,而SCCN03株RdRp的延伸链和无规则卷曲比例比SCH04株略高,分别为12.79%和43.41%;SCCN03株RdRp比SCH04株多4个磷酸化位点和1个N-糖基化位点。分析结果表明不同型间RHDV RdRp基因核苷酸变异率较高,GI.1 RHDV毒株间的变异范围大于GI.2 RHDV毒株。SCH04株和SCCN03株RHDV RdRp的氨基酸序列相似性高于核苷酸序列相似性,其二级结构和高级结构差异较小,遗传相对稳定。本研究为RHDV的遗传演变和生物合成等分子病原学研究提供了科学参考。

Small Amplicons Mutation Library for Vaccine Screening by Error-Prone Polymerase Chain Reaction

Library construction is a common method used to screen target genes in molecular biology.Most library constructions are not suitable for a small DNA library(<100 base pair(bp))and low RNA library output.To maximize the library’s complexity,error-prone polymerase chain reaction(PCR)was used to increase the base mutation rate.After introducing the DNA fragments into the competent cell,the library complexity could reach 109.Library mutation rate increased exponentially with the dilution and amplification of error-prone PCR.The error-prone PCR conditions were optimized including deoxyribonucleotide triphosphate(dNTP)concentration,Mn^(2+)concentration,Mg^(2+)concentration,PCR cycle number,and primer length.Then,a RNA library with high complexity can be obtained by in vitro transcription to meet most molecular biological screening requirements,and can also be used for mRNA vaccine screening.

Deoxyribonucleic Acid-Polymerase Chain Reaction Status of HIV Exposed Infants in a Sub Regional Prevention of Mother-to-Child Transmission of HIV Programme during the Period 2009-2020

Introduction: Transitioning to more efficacious Antiretrovirals for HIV infected pregnant women and infant prophylaxis has reduced Mother to child transmission of HIV significantly. This study aimed to determine HIV infection status in HIVexposed infants who had their first DNA polymerase chain reaction test in our molecular Laboratory. Subjects, Materials and Methods: Dried Blood Spots for HIV DNA results from 5 states between 2009 and 2020 were analyzed in the PCR laboratory of the Federal Teaching Hospital, Gombe. Results: Nine thousand eight hundred and twenty-three Human Immunodeficiency Virus Deoxyribonucleic acid polymerase Chain Reaction results were analysed;4937 (50.2%) were males. During the study period, there was an overall declining trend in the mother-to-child transmission rate from 3.8% in 2009 to 1.0% in 2020. 6120 (62.3%) of HIV + mothers received Highly active antiretroviral therapy HAART before pregnancy. 7845 (76.2%) of the infants received Nevirapine prophylaxis. Dried blood spot samples were collected from 4077 (41.5%) at 6 - 8 weeks. 8438 (85.9%) received cotrimoxazole. 9469 (96.4%) were ever breastfed. Of the 9823 HIV DNA PCR results, 255 (2.6%) were positive while 69/4077 (1.7%) and 109/2662 (4.1%) were positive for HIV DNA at 6 - 8 weeks and > 12 weeks respectively. (p = 0.001). 86/747 (11.5%) of infants whose HIV-positive mothers received no ARVS were HIV DNA positive. (p = 0.001). 106/884 (12.0%) of infants who had no Antiretroviral prophylaxis had positive HIV DNA results;7/413 (1.7%) with Zidovudine/Nevirapine prophylaxis had positive results. (p = 0.001). 246/9469 (2.6%) of infants that were ever breastfed were positive for HIV DNA;11/354 (3.0%) that never breastfed had positive HIV DNA. Conclusion: Lack of maternal/infant ARVs and prolonged breastfeeding increased the risk of infant HIV infection.

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections,rt269L and rt269I

BACKGROUND The presence of two distinct hepatitis B virus(HBV)Pol RT polymorphisms,rt269L and rt269I,could contribute to the unique clinical or virological phenotype of HBV genotype C2.Therefore,a simple and sensitive method capable of identifying both types in chronic hepatitis B(CHB)patients infected with genotype C2 should be developed.AIM To develop a novel simple and sensitive locked nucleic acid(LNA)-real timepolymerase chain reaction(RT-PCR)method capable of identifying two rt269 types in CHB genotype C2 patients.METHODS We designed proper primer and probe sets for LNA-RT-PCR for the separation of rt269 types.Using synthesized DNAs of the wild type and variant forms,melting temperature analysis,detection sensitivity,and endpoint genotyping for LNA-RT-PCR were performed.The developed LNA-RT-PCR method was applied to a total of 94 CHB patients of genotype C2 for the identification of two rt269 polymorphisms,and these results were compared with those obtained by a direct sequencing protocol.RESULTS The LNA-RT-PCR method could identify two rt269L and rt269I polymorphisms of three genotypes,two rt269L types[‘L1’(WT)and‘L2’]and one rt269I type(‘I’)in single(63 samples,72.4%)or mixed forms(24 samples,27.6%)in 87(92.6%sensitivity)of 94 samples from Korean CHB patients.When the results were compared with those obtained by the direct sequencing protocol,the LNA-RT-PCR method showed the same results in all but one of 87 positive detected samples(98.9%specificity).CONCLUSION The newly developed LNA-RT-PCR method could identify two rt269 polymorphisms,rt269L and rt269I,in CHB patients with genotype C2 infections.This method could be effectively used for the understanding of disease progression in genotype C2 endemic areas.

Confusing finding of quantitative fluorescent polymerase chain reaction analysis in invasive prenatal genetic diagnosis:A case report

BACKGROUND Quantitative fluorescent polymerase chain reaction(QF-PCR)is a rapid prenatal diagnostic method for abnormalities on chromosomes 21,18,and 13 and sex chromosomal aneuploidy.However,the value of QF-PCR in diagnosing chromosomal structural abnormalities is limited.In this article,we report a confusing QF-PCR finding in a pregnant woman who underwent amniocentesis.CASE SUMMARY The short tandem repeat marker AMXY(Xp22.2/Yp11.2)located on the sex chromosome exhibited a trisomic biallelic pattern,indicating that the karyotype of the fetus might be 47,XYY.Chromosome analysis performed on cultured amniocytes showed a normal male karyotype of the fetus.Copy number variation sequencing confirmed a 500 kb duplication at Yp11.2-Yp11.2(chrY:6610001_7110000)and a 250 kb duplication at Yp11.2-Yp11.2(chrY:7110001_7360000).CONCLUSION In conclusion,the comprehensive application of different methods could achieve a higher detection rate and accuracy for the prenatal diagnosis of chromosomal disorders through chromosomal testing.

Establishment of a rapid detection method of Ureaplasma urealyticum based on recombinant polymerase amplification

Ureaplasma urealyticum(UU),is one of the most vital pathogens causing genitourinary tract infections of the body,and it can result in poor maternal and perinatal outcomes.The aim of this study was to establish a method to detect Ureaplasma urealyticum based on recombinant polymerase amplification(RPA)technique.Specific primers and probes were designed according to the 16sRNA gene sequence of Ureaplasma urealyticum.Six pathogens were detected for real-time fluorescence RPA specificity verification,including Mycoplasma hominis(MH),Chlamydia trachomatis(CT),Neisseria gonorrhoeae(NG),Staphylococcus aureus,Escherichia coli,and Lactobacillus vaginalis.The sensitivity of the method was performed by gradient dilution of the extracted template.A total of 60 clinical samples were detected by the established real-time fluorescence RPA.Detection of Ureaplasma urealyticum can be completed within 20 minutes at 39°C using established RPA method.The minimum detection limit of Ureaplasma urealyticum by real-time fluorescence RPA was 3 pg.The evaluation of 60 clinical samples proved that RPA method was feasible.A high specificity,sensitivity,simplicity and rapidity method for Ureaplasma urealyticum detection was successfully established based on the real-time fluorescence RPA method.

Taq DNA聚合酶的分子改造及其在探针法qPCR直扩体系中的应用

Taq DNA聚合酶作为实时荧光定量聚合酶链式反应(qPCR)技术的核心组分,其性能优劣直接影响qPCR技术的进一步发展。然而,野生型Taq DNA聚合酶的耐抑制剂性能差、延伸性能不足。为获得具有高性能的Taq DNA聚合酶,采用基因工程技术将双链DNA结合蛋白Sso7d或Sto7d融合在野生型Taq DNA聚合酶的N端或C端,构建了4个均可溶表达的改造体,再经过耐受性测试筛选较优的改造体,结果显示:改造体Taq-Sto的耐受性最高,其热稳定性不受影响,且在1 s/kbp的延伸条件下能成功扩增靶标,表明Taq-Sto具有增强的延伸性能,在TaqMan探针法qPCR体系中对腐殖酸、单宁酸、全血等抑制剂同样表现出良好的耐受性。EMSA实验发现:Taq-Sto对DNA模板的结合亲和力有所提高,有利于增强Taq-Sto对模板的竞争力;将Taq-Sto应用于非洲猪瘟病毒(ASFV)的TaqMan探针法qPCR检测,与商品化试剂相比,Taq-Sto具有更低的ASFV检出限,且在体积分数为2%~6%的猪粪便样本或猪肉样本中的检测灵敏度分别为100.0%和85.4%,说明Taq-Sto在直扩qPCR检测领域更具有优势。

快速检测十足目虹彩病毒1 RPA-LFD方法的建立及初步应用

为建立一种高效、快速、简便的十足目虹彩病毒1(DIV1)检测方法,本研究以DIV1 ATPase基因为检测靶标,设计特异性引物和探针,采用方阵法优化反应温度和时间,结果显示,重组酶聚合酶扩增技术(RPA)最优反应温度为37℃,时间为15 min,结果观察时间为5 min,检测过程总时长为20 min,初步建立了DIV1的重组酶聚合酶扩增技术结合侧向流试纸条方法(RPA-LFD)。提取其他7种常见虾类病原基因组与DIV1基因组,采用该方法检测,分析其特异性;构建重组质粒标准品p UC57-DIV1,10倍倍比稀释后采用该方法检测,分析其灵敏性;以3种不同浓度的重组质粒标准品为模板进行批间、批内重复性试验。结果显示,该方法能特异性检测DIV1,与虾类其他常见易感病原均无交叉反应;其对重组质粒标准品的检测限为1×10^(1)拷贝/μL;批内和批间重复性试验的检测结果均一致,重复性好。利用该方法与已发表的荧光定量PCR(qPCR)同时检测15份感染DIV1的罗氏沼虾样品及40份临床样品,结果显示,该方法的阳性检测率为69.09%(38/55),阴性率为30.91%(17/55),与q PCR检测结果一致,二者的阳性符合率、阴性符合率、总符合率均为100%。综上所述,本研究建立的RPA-LFD方法检测DIV1具有快速、简便、灵敏度高且特异性强的特点,且不需要精密昂贵的仪器设备,为基层实验室及现场检测提供了可行技术手段。

4种植物源性成分多重real-time PCR检测方法的建立及其在食用淀粉中的应用

建立一种可同时快速检测红薯、木薯、马铃薯、玉米源性成分的多重实时聚合酶链式反应(real-time polymerase chain reaction,real-time PCR)方法。分别以红薯g3pdh基因、木薯g3pdh基因、马铃薯UGPase基因、玉米zSSIIb基因为靶基因设计特异性引物和TaqMan探针,以18S rRNA基因为内参基因,建立多重real-time PCR方法,开展方法学验证,并对不同掺入比例模拟样品和实际淀粉样品进行检测。结果显示,该方法具有高通量、特异性强、灵敏度高等优点。与15种非目标源性均无交叉反应;对目标DNA的检测灵敏度可达到3×10^(-3) ng/μL,且具有良好的线性关系和扩增效率;对淀粉样品的检出限可达0.1%,对50份实际样品进行检测,结果与参比方法一致,说明建立的多重real-time PCR法可用于食用淀粉种类掺假鉴别检测。

等温扩增技术在水产品寄生虫检测中的应用研究进展

水产品中寄生虫可引起食源性疾病,已成为影响食品安全的重要因素之一。近年来,基于传统分子生物学发展而来的等温扩增技术以其恒温、高效、耗时短、不过度依赖设备和仪器等优点,逐渐应用于分子诊断和疾病检测。该文在介绍等温扩增技术原理与特点的基础上,综述了其在水产品中寄生虫检测方面的应用研究进展,提出存在问题与解决办法,并展望了等温扩增技术的应用前景,以期为快检技术在水产品质量安全控制技术中的研究开发提供理论参考。

土壤固碳微生物的绝对定量检测实验设计

为了定量检测土壤中的固碳微生物,设计以功能基因cbbL为靶标的固碳微生物微滴数字聚合酶链式反应(ddPCR)检测方法。选择合适的引物探针,从退火温度、探针浓度以及引物浓度进行反应条件的优化,分析ddPCR检测方法的线性范围、敏感性、重复性和特异性。结果显示,当退火温度为55.8℃、探针与引物浓度分别为350、750 nmol/L时,建立的cbbL-ddPCR扩增反应效率最高,阴阳性微滴分布界限最明显,平均拷贝数较高;检测的线性范围为2.3×10^(0)~2.3×10^(5)copies/μL-DNA,曲线方程y=0.1077x-95.562,相关系数R^(2)为0.9997,检出限为0.5 copy/μL-DNA,21个重复的变异系数仅为3.92%,与其他4种非固碳微生物DNA未发生交叉反应。所建立的cbbL-ddPCR方法可用于土壤微生物固碳潜能测定。

多重PCR毛细管电泳细菌快速鉴定方法的建立和临床应用研究

目的针对引起人类感染的常见病原菌建立一种基于多重PCR毛细管电泳技术的快速细菌鉴定方法,评估其临床应用价值。方法建立23种常见病原菌的多重PCR毛细管电泳检测体系。收集150例临床微生物检测标本,分别用多重PCR毛细管电泳法(以下简称多重PCR法)、培养法进行检测。对两种方法的检测结果进行比较,评价多重PCR法的检测效能。结果150例标本中多重PCR法检出15种病原菌、培养法检出14种病原菌。多重PCR法检测阳性率为73.3%,培养法为70.0%,差异无统计学意义(P>0.05)。多重PCR法对肺炎链球菌的检出率为16.0%,培养法为6.0%,差异有统计学意义(P<0.05)。多重PCR法对混合菌标本的检出率为15.3%,培养法未检出混合菌标本。多重PCR法与培养法检测结果的符合率为79.3%。多重PCR法检测时间为3~6 h,培养法为2~4 d。结论与培养法比较,多重PCR法具有较高的时效性,对肺炎链球菌及混合菌标本具有较高的检出率,可满足临床标本病原微生物快速初筛的需求。

miRNA表达谱在产前诊断胎儿先天性心脏病中的研究

目的:探究微小RNA(microRNA,miRNA)表达谱在产前诊断胎儿先天性心脏病(congenital heart disease,CHD)中的应用。方法:收集2021年1月—2022年12月于天津市中心妇产科医院就诊的30例超声确诊为CHD的孕妇(病例组)和同期10例要求行羊水穿刺的孕妇(对照组),用Illumina测序平台对2组孕妇的羊水上清进行全转录组测序,2组孕妇的全部miRNA进行归一化,分析差异表达的miRNA。从差异表达的miRNA中挑选P<0.05和|log2 FC|>3(差异倍数,Fold Change,FC)的miRNA再在羊水和外周血中进行实时荧光定量聚合酶链反应(real time fluorescence quantitative polymerase chain reaction,RT-qPCR)验证,比较羊水中miRNA测序与RT-qPCR的差异倍数,挑选外周血与羊水表达调控方向一致的miRNA。结果:共发现138个差异表达miRNA,其中85个上调,53个下调。进一步挑选出了15个差异表达的miRNA,羊水中miRNA测序与RT-qPCR结果比较相一致。外周血与羊水中表达调控方向一致的miRNA有2个,分别为miR-222-3p和miR-189-5p,这2个miRNA在病例组母血中表达量较对照组显著上升(均P<0.05)。结论:母血中miRNA作为新的血清学标志物可以初步应用于筛查胎儿CHD。

基于主成分分析的多重定量PCR荧光串扰校正

聚合酶链式反应(PCR)是分子生物学常用的检测手段,主要用于对生物的DNA或RNA进行检测。由于荧光光谱重叠和滤光片过滤带宽限制,检测时所获得的荧光数据通常会包含荧光通道之间的串扰,串扰的存在使PCR结果分析变得复杂,并可能影响最终的检测结果。选择合适的光学元件,并确定通道间的补偿矩阵,可以降低甚至消除荧光串扰。目前荧光补偿矩阵大多通过迭代计算获得,还没有一种简单的方法可以从混合的多通道荧光数据中找到荧光补偿矩阵。为了快速获得荧光补偿矩阵,减小计算量,采用主成分分析法(PCA)中确定主成分的方式,基于搭建的测试平台进行单一染料实验,获得染料的荧光信号在各个检测通道的分布情况,计算得到荧光补偿矩阵。通过分析补偿矩阵,发现对于搭建的硬件系统,Cy5染料对Cy5.5通道串扰较大,串扰比例为8.76%,同时Cy5.5染料对Cy5通道串扰影响也相对较大,比例约为6.2%;其次是ROX染料对HEX通道串扰,比例约为2.68%;HEX染料对FAM通道串扰,比例约为1.58%;FAM染料对HEX通道串扰相对较小,比例约为0.25%,其余通道无明显串扰,与荧光光谱反映的结果一致。采用得到的荧光补偿矩阵对单一染料实验得到的原始荧光数据进行处理,有效去除了非目标通道的荧光串扰,实现了荧光通道数据的解耦,验证了方法的可行性。最后设计了染料颜色分辨实验,将不同浓度的多种染料进行组合测试,并采用所提出的方法将得到的数据进行荧光补偿。实验结果表明,荧光通道各自的线性相关性较高,五个荧光通道的线性相关系数r均大于0.99,该结果进一步验证了该补偿方法的有效性。

细菌转录翻译偶联机制研究

中心法则描述了遗传信息从DNA到RNA再到蛋白质的传递过程。转录过程是RNA聚合酶以DNA为模板合成信使RNA的过程,翻译过程是核糖体利用信使RNA合成蛋白质的过程。与真核生物不同,细菌和古菌没有细胞核膜的分隔,其转录过程和翻译过程在相同时间、相同位置上进行。其中,RNA聚合酶与核糖体相互协同,同步完成转录和翻译的现象被称为转录翻译偶联。转录翻译偶联是细菌和古菌的一种重要基因调控机制,能同时有效地调控转录过程和翻译过程,是细菌适应复杂环境的重要生物学基础。数十年来,大量的研究逐步揭示了细菌转录翻译偶联机制在细菌基因表达调控中的作用,一系列参与转录翻译偶联过程的调控因子也被鉴定发现。近期,基于不同偶联状态的转录翻译偶联复合体结构的突破性研究,首次系统地展示了在不同信使RNA间距下,转录翻译偶联过程的动态变化,为后续研究转录翻译偶联基因调控机制提供了理论基础。

PARP抑制剂与免疫检查点抑制剂联合治疗在妇科恶性肿瘤中的应用

近年来肿瘤靶向和免疫治疗的研究进展迅速,如多腺苷二磷酸核糖聚合酶抑制剂[poly(ADP-ribose)polymerase inhibitor,PARPi]、免疫检查点抑制剂(immune checkpoint inhibitors,ICI)等已改变了妇科肿瘤的传统治疗模式,但部分患者疗效有限或出现耐药。临床前研究发现,PARPi损伤DNA修复过程,可造成肿瘤突变负荷与肿瘤特异性抗原增加,调节肿瘤微环境,刺激肿瘤浸润淋巴细胞(tumor infiltrating lymphocytes,TIL)产生并促进抗肿瘤免疫反应,为PARPi与ICI联合治疗提供了理论基础。近年多项临床研究发现PARPi与ICI联合使用可显著改善妇科恶性肿瘤患者的预后。