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Real-time Quantification of nuclear RNA export using an intracellular relocation probe

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Chinese Chemical Letters 2022年第8期33卷 3865-3868页

作者： Jie Shen Juan Chen Dong Wang Zhengjie Liu Guangmei Han Bianhua Liu Mingyong Han Ruilong Zhang Guodong Liu Zhongping Zhang School of Chemistry and Chemical Engineering Information Materials and Intelligent Sensing Laboratory of Anhui Provinceand Institute of Physical Science and Information TechnologyAnhui UniversityHefei 230601China Key Laboratory of Structure and Functional Regulation of Hybrid Materials(Anhui University) Ministry of EducationHefei 230601China Key Lab of Photovoltaic and Energy Conservation Materials Institute of Solid State PhysicsHFIPSChinese Academy of SciencesHefei 230031China School of Life and Health Sciences Anhui Science and Technology UniversityChuzhou 233100China

Nuclear RNA export into the cytoplasm is one of the key steps in protein expression to realize biological functions.Despite the broad availability of nucleic acid dyes,tracking and quantifying the highly dynamic process of RNA export in live cells is challenging.When dye-labeled RNA enters the cytoplasm,the dye molecules are released upon degradation of the RNA,allowing them to re-enter the cell nucleus.As a result,the ratio between the dye exported with RNA into the cytoplasm and the portion staying inside the nucleus cannot be determined.To address this common limitation,we report the design of a smart probe that can only check into the nucleus once.When adding to cells,this probe rapidly binds with nuclear RNAs in live cells and reacts with intrinsic H_(2)S.This reaction not only activates the fluorescence for RNA tracking but also changes the structure of probe and consequently its intracellular localization.After disassociating from exported RNAs in cytoplasm,the probe preferentially enters lysosomes rather than cell nucleus,enabling real-time quantitative measurement of nuclear RNA exports.Using this probe,we successfully evaluated the effects of hormones and cancer drugs on nuclear RNA export in live cells.Interestingly,we found that hormones inhibiting RNA exports can partially offset the effect of chemotherapy.

关键词： RNA export Quantification Relocation probe Fluorescence imaging Hormone

GC-NICI-MS analysis of acetazolamide and other sulfonamide (R-SO2-NH2)drugs as pentafluorobenzyl derivatives [R-SO2-N(PFB)2] and Quantification of pharmacological acetazolamide in human urine

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Journal of Pharmaceutical Analysis 2020年第1期10卷 49-59页

作者： Olga Begou Kathrin Drabert Georgios Theodoridis Dimitrios Tsikas Institute of Toxicology Core Unit ProteomicsHannover Medical SchoolCarl-Neuberg-Strasse 1D-30625HannoverGermany Department of Chemistry Aristotle University of Thessaloniki54124ThessalonikiGreece BIOMIC_AUTh Center for Interdisciplinary Research and Innovation(CIRI-AUTH)Balkan Center10th Km Thessaloniki-Thermi RdP.O.Box 8318GR 57001ThessalonikiGreece

Acetazolamide(molecular mass(MM),222)belongs to the class of sulfonamides(R-SO2-NH2)and is one of the strongest pharmacological inhibitors of carbonic anhydrase activity.Acetazolamide is excreted unchanged in the urine.Here,we report on the development,validation and biomedical application of a stable-isotope dilution GC-MS method for the reliable quantitative determination of acetazolamide in human urine.The method is based on evaporation to dryness of 50 mL urine aliquots,base-catalyzed derivatization of acetazolamide(d0-AZM)and its internal standard[acetylo-2H3]acetazolamide(d3-AZM)in 30 vol%pentafluorobenzyl(PFB)bromide in acetonitrile(60 min,30C),reconstitution in toluene(200 mL)and injection of 1-mL aliquots.The negative-ion chemical ionization(NICI)mass spectra(methane)of the PFB derivatives contained several intense ions including[M]‒at m/z 581 for d0-AZM and m/z 584 for d3-AZM,suggesting derivatization of their sulfonamide groups to form N,N-dipentafluorobenzyl derivatives(R-SO2-N(PFB)2),i.e.,d0-AZM-(PFB)2 and d3-AZM-(PFB)2,respectively.Quantification was performed by selected-ion monitoring of m/z 581 and 83 for d0-AZM-(PFB)2 and m/z 584 and 86 for d3-AZM-(PFB)2.The limits of detection and quantitation of the method were determined to be 300 fmol(67 pg)and 1 mM of acetazolamide,respectively.Intra-and inter-assay precision and accuracy for acetazolamide in human urine samples in pharmacologically relevant concentration ranges were determined to be 0.3%e4.2%and 95.3%e109%,respectively.The method was applied to measure urinary acetazolamide excretion after ingestion of a 250 mg acetazolamide-containing tablet(Acemit®)by a healthy volunteer.Among other tested sulfonamide drugs,methazolamide(MM,236)was also found to form a N,N-dipentafluorobenzyl derivative,whereas dorzolamide(MM,324)was hardly detectable.No GC-MS peaks were obtained from the PFB bromide derivatization of hydrochlorothiazide(MM,298),xipamide(MM,355),indapamide and metholazone(MM,366 each)or brinzolamide(MM,384).We demonstrate for the first

关键词： Acetazolamide Derivatization Quantification Sulfonamides Validation

CircAST:Full-length Assembly and Quantification of Alternatively Spliced Isoforms in Circular RNAs

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Genomics, Proteomics & Bioinformatics 2019年第5期17卷 522-534页

作者： Jing Wu Yan Li Cheng Wang Yiqiang Cui Tianyi Xu Chang Wang Xiao Wang Jiahao Sha Bin Jiang Kai Wang Zhibin Hu Xuejiang Guo Xiaofeng Song Department of Biomedical Engineering Nanjing University of Aeronautics and AstronauticsNanjing 211106China School of Biomedical Engineering and Informatics Nanjing Medical UniversityNanjing 211166China State Key Laboratory of Reproductive Medicine Nanjing Medical UniversityNanjing 211166China Center of Pathology and Clinical Laboratory Sir Run Run HospitalNanjing Medical UniversityNanjing 211166China Center for Cellular and Molecular Therapeutic Children’s Hospital of PhiladelphiaPhiladelphiaPA 19104USA

Circular RNAs(circ RNAs),covalently closed continuous RNA loops,are generated from cognate linear RNAs through back splicing events,and alternative splicing events may generate different circ RNA isoforms at the same locus.However,the challenges of reconstruction and Quantification of alternatively spliced full-length circ RNAs remain unresolved.On the basis of the internal structural characteristics of circ RNAs,we developed Circ AST,a tool to assemble alternatively spliced circ RNA transcripts and estimate their expression by using multiple splice graphs.Simulation studies showed that Circ AST correctly assembled the full sequences of circ RNAs with a sensitivity of 85.63%–94.32%and a precision of 81.96%–87.55%.By assigning reads to specific isoforms,Circ AST quantified the expression of circ RNA isoforms with correlation coefficients of 0.85–0.99 between theoretical and estimated values.We evaluated Circ AST on an in-house mouse testis RNA-seq dataset with RNase R treatment for enriching circ RNAs and identified 380 circ RNAs with full-length sequences different from those of their corresponding cognate linear RNAs.RT-PCR and Sanger sequencing analyses validated 32 out of 37 randomly selected isoforms,thus further indicating the good performance of Circ AST,especially for isoforms with low abundance.We also applied Circ AST to published experimental data and observed substantial diversity in circular transcripts across samples,thus suggesting that circ RNA expression is highly regulated.Circ AST can be accessed freely at https://***/xiaofengsong/CircAST.

关键词： Circular RNA Full-length reconstruction Isoform Quantification Multiple splice graph model Transcriptome

Development of a fast and precise method for simultaneous Quantification of the PLGA monomers lactic and glycolic acid by HPLC

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Journal of Pharmaceutical Analysis 2019年第2期9卷 100-107页

作者： Marcel Pourasghar Aljoscha Koenneke Peter Meiers Marc Schneider Department of Pharmacy Biopharmaceutics and Pharmaceutical TechnologySaarland University

Poly(lactide-co-glycolide acid)(PLGA) is an extraordinary well-described polymer and has excellent pharmaceutical properties like high biocompatibility and good biodegradability. Hence, it is one of the most used materials for drug delivery and biomedical systems, also being present in several US Food and Drug Administration-approved carrier systems and therapeutic devices. For both applications, the Quantification of the polymer is inalienable. During the development of a production process, parameters like yield or loading efficacy are essential to be determined. Although PLGA is a well-defined biomaterial,it still lacks a sensitive and convenient Quantification approach for PLGA-based systems. Thus, we present a novel method for the fast and precise Quantification of PLGA by RP-HPLC. The polymer is hydrolyzed into its monomers, glycolic acid and lactic acid. Afterwards, the monomers are derivatized with the absorption-enhancing molecule 2,4′-dibromoacetophenone. Furthermore, the wavelength of the derivatized monomers is shifted to higher wavelengths, where the used solvents show a lower absorption,increasing the sensitivity and detectability. The developed method has a detection limit of 0.1 mg/mL,enabling the Quantification of low amounts of PLGA. By quantifying both monomers separately, information about the PLGA monomer ratio can be also directly obtained, being relevant for degradation behavior. Compared to existing approaches, like gravimetric or nuclear magnetic resonance measurements, which are tedious or expensive, the developed method is fast, ideal for routine screening, and it is selective since no stabilizer or excipient is interfering. Due to the high sensitivity and rapidity of the method, it is suitable for both laboratory and industrial uses.

关键词： Poly(lactide-co-glycolide acid)(PLGA) Alkaline hydrolysis Quantification Drug delivery system RP-HPLC

QuaPra: Efficient transcript assembly and Quantification using quadratic programming with Apriori algorithm

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Science China(Life Sciences) 2019年第7期62卷 937-946页

作者： Xiangjun Ji Weida Tong Baitang Ning Christopher E. Mason David P. Kreil Pawel P. Labaj Geng Chen Tieliu Shi The Center for Bioinformatics and Computational Biology Shanghai Key Laboratory of Regulatory BiologyInstitute of Biomedical Sciences and School of Life SciencesEast China Normal UniversityShanghai 200241China National Center for Toxicological Research U.S. Food and Drug Administration3900 NCTR RDJefferson AR 72079USA Department of Physiology and Biophysics Weill Cornell MedicineNew York 10065USA The HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine New York 10021USA Feil Family Brain & Mind Research Institute New York 10065USA Chair of Bioinformatics Research Group Boku UniversityVienna A-1190Austria Malopolska Centre of Biotechnology Jagiellonian UniversityKrakow 30-387Poland APART Fellow Austrian Academy of ScienceVienna A-1190Austria National Center for International Research of Biological Targeting Diagnosis and Therapy Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy ResearchCollaborative Innovation Center for Targeting Tumor Diagnosis and TherapyGuangxi Medical UniversityNanning 530021China

RNA sequencing(RNA-seq) has greatly facilitated the exploring of transcriptome landscape for diverse organisms.However,transcriptome reconstruction is still challenging due to various limitations of current tools and sequencing technologies.Here,we introduce an efficient tool,QuaPra(Quadratic Programming combined with Apriori),for accurate transcriptome assembly and Quantification.QuaPra could detect at least 26.5% more low abundance(0.1–1 FPKM) transcripts with over 2.7% increase of sensitivity and precision on simulated data compared to other currently popular tools.Moreover,around one-quarter more known transcripts were correctly assembled by QuaPra than other assemblers on real sequencing data.QuaPra is freely available at http://***/QuaPra/.

关键词： RNA-Seq transcriptome reconstruction transcript assembly transcript Quantification

Quantification of skid resistance seasonal variation in asphalt pavements

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Journal of Traffic and Transportation Engineering(English Edition) 2020年第2期7卷 237-248页

作者： Christina Plati Maria Pomoni Konstantina Georgouli Laboratory of Pavement Engineering School of Civil EngineeringNational Technical University of Athens(NTUA)Athens 15773Greece

The present study deals with the Quantification of skid resistance seasonal variation in asphalt pavements.Consequently,skid resistance data were collected twice per year(after"wet"and"dry"seasons)along four trial sections of an urban highway with a GripTester system(during a six-year field experiment).For the estimation of a percentage reference change(%)between wet and dry skid resistance data,a methodology is developed based on distribution fitting of the percentage change of skid resistance index due to seasonal variation impact.The outcome of the analysis is applied and successfully validated through a set of skid resistance data collected from an interurban highway pavement section with different traffic and environmental conditions.As such the developed methodology can be considered as a promising tool for accommodating the seasonal variation of skid resistance.Further,the resulting percentage reference change(%)enables the estimation of skid resistance level for the dry period based on the wet period measurements.Overall,it seems that the proposed Quantification process may constitute a practical yet efficient tool for road authorities and highway operators on how to estimate the effect of seasonal variation on skid resistance levels and schedule well-ahead necessary actions for enhancing driving safety.

关键词： Asphalt pavement Skid resistance Seasonal variation Quantification Road safety

Uncertainty Quantification for stochastic dynamical systems using time-dependent stochastic bases

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Applied Mathematics and Mechanics(English Edition) 2019年第1期40卷 63-84页

作者： Jinchun LAN Qianlong ZHANG Sha WEI Zhike PENG Xinjian DONG Wenming ZHANG State Key Laboratory of Mechanical System and Vibration School of Mechanical EngineeringShanghai Jiao Tong University

A novel method based on time-dependent stochastic orthogonal bases for stochastic response surface approximation is proposed to overcome the problem of significant errors in the utilization of the generalized polynomial chaos(GPC) method that approximates the stochastic response by orthogonal polynomials. The accuracy and effectiveness of the method are illustrated by different numerical examples including both linear and nonlinear problems. The results indicate that the proposed method modifies the stochastic bases adaptively, and has a better approximation for the probability density function in contrast to the GPC method.

关键词： uncertainty Quantification stochastic response surface approximation timedependent orthogonal bases polynomial chaos

Value of elastography point Quantification in improving the diagnostic accuracy of early diabetic kidney disease

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World Journal of Clinical Cases 2019年第23期7卷 3945-3956页

作者： Qiu-Yun Liu Qi Duan Xiao-Hong Fu Li-Qian Fu Hong-Wei Xia Yong-Lin Wan Department of Ultrasound Naval Military Medical University Affiliated Gongli HospitalShanghai 200135Chi Department of Ultrasound Shanghai Hemujia HospitalShanghai 200336China Department of Nephrology Naval Military Medical University Affiliated Gongli HospitalShanghai 200135China

BACKGROUND Diabetic kidney disease(DKD)is a common complication of diabetes.The patient’s prognosis is poor once DKD progresses to advanced stage.Accurate diagnosis and timely treatment of early DKD are important for improving patient’s prognosis and reducing mortality.AIM To explore the value of elastography point Quantification(ElastPQ)in improving the accuracy of early DKD diagnosis.METHODS A total of 69 patients with type 2 diabetes were recruited from Naval Military Medical University Affiliated Gongli Hospital.Patients were divided into early DKD group and medium DKD group according to pathological results and urinary albumin excretion rate(UAER).Another 40 patients with simple diabetes were included as the diabetes group.The baseline data,laboratory diagnostic indicators,and ultrasound indicators for each patient were recorded.The differences of the indicators in the three groups were compared.Multivariate logistic regression was used to analyze the influencing factors of the development from simple diabetes into early DKD and from early DKD into medium DKD.Receiver operating characteristic analyses of potential indicators in identifying early DKD and medium DKD,and early DKD and simple diabetes were established.RESULTS Multivariate logistic regression analysis showed that UAER(P0.9].The accuracy of UAER(AUC=0.744),diabetes duration(AUC=0.757),and renocortical YM(AUC=0.782)for the diagnosis of early DKD and simple diabetes were limited.However,the combined diagnosis of UAER,diabe

关键词： Diabetic kidney disease Elastography point Quantification Young's Modulus Urinary albumin excretion rate Combined diagnosis Diabetes

Evaluation of real-time RT-PCR assays for detection and Quantification of norovirus genogroups Ⅰ and Ⅱ

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Virologica Sinica 2017年第2期32卷 139-146页

作者： Kitwadee Rupprom Porntip Chavalitshewinkoon-Petmitr Pornphan Diraphat Leera Kittigul Department of Microbiology Faculty of Public Health Mahidol University Bangkok 10400 Thailand Department of Protozoology Faculty of Tropical Medicine Mahidol University Bangkok 10400 Thailand

Noroviruses are the leading cause of acute gastroenteritis in humans. Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) is a promising molecular method for the detection of noroviruses. In this study, the performance of three Taq Man real-time RT-PCR assays was assessed, which were one commercially available real-time RT-PCR kit(assay A:Norovirus Real Time RT-PCR kit) and two in-house real-time RT-PCR assays(assay B: Light Cycler RNA Master Hybprobe and assay C: Real Time ready RNA Virus Master). Assays A and B showed higher sensitivity than assay C for norovirus GI, while they all had the same sensitivity(103 DNA copies/m L) for GII DNA standard controls. Assay B had the highest efficiency for both genogroups.No cross-reactivity was observed among GI and GII noroviruses, rotavirus, hepatitis A virus, and poliovirus. The detection rates of these assays in GI and GII norovirus-positive fecal samples were not significantly different. However, the mean Quantification cycle(Cq) value of assay B for GII was lower than assays A and C with statistical significance(P-value, 0.000). All three real-time RT-PCR assays could detect a variety of noroviruses including GI.2, GII.2, GII.3, GII.4, GII.6, GII.12, GII.17,and GII.21. This study suggests assay B as a suitable assay for the detection and Quantification of noroviruses GI and GII due to good analytical sensitivity and higher performance to amplify norovirus on DNA standard controls and clinical samples.

关键词： norovirus genogroup real-time RT-PCR Quantification