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Novel degradation products of argatroban: Isolation, synthesis and extensive characterization using NMR and LC-PDA-MS/Q-TOF

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Journal of Pharmaceutical Analysis 2018年第2期8卷 86-95页

作者： Vinodh Guvvala Venkatesan Chidambaram Subramanian jaya shree anireddy Mahesh Konda Centre for Chemical Science&Technology Institute of Science& Technology JNTUH Gland Pharma Ltd. Research and Development D.P.Pally

Forced degradation study of argatroban under conditions of hydrolysis(neutral, acidic and alkaline), oxidation,photolysis and thermal stress, as suggested in the ICH Q1 A(R2), was accomplished. The drug showed significant degradation under hydrolysis(acidic, alkaline) and oxidation(peroxide stress) conditions. The drug remained stable under thermal and photolytic stress conditions. In total, seven novel degradation products(DP-1 to DP-7) were found under diverse conditions, which were not reported earlier. The chemical structures of these degradation products were characterized by ~1H NMR,^(13)C NMR, 2 D NMR, Q-TOF-MSnand IR spectral analysis and the proposed degradation products structures were further confirmed by the individual synthesis.

关键词： Argatroban Forced degradation NMR LC-PDA LC-MS/Q-TOF-MSn

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Phytophthora Effectors Modulate Genome-wide Alternative Splicing of Host mRNAs to Reprogram Plant Immunity

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Molecular Plant 2020年第10期13卷 1470-1484页

作者： Jie Huang Xinyu Lu Hongwei Wu Yuchen Xie Qian Peng Lianfeng Gu Juyou Wu Yuanchao Wang anireddy SNReddy Suomeng Dong Department of Plant Pathology Nanjing Agricultural UniversityNanjing 210095China Basic Forestry and Proteomics Research Center College of ForestryFujian Agriculture and Forestry UniversityFuzhou 350002China State Key Laboratory of Crop Genetics and Germplasm Enhancement College of HorticultureNanjing Agricultural UniversityNanjing 210095China Colorado State University Program in Cell and Molecular BiologyFort CollinsCO 80523USA Key Laboratory of Integrated Management of Crop Diseases and Pests(Ministry of Education) Nanjing 210095China The Key Laboratory of Plant Immunity Nanjing Agricultural UniversityNanjing 210095China

Alternative splicing(AS)of pre-mRNAs increases transcriptome and proteome diversity,regulates gene expression through multiple mechanisms,and plays important roles in plant development and stress ***,the prevalence of genome-wide plant AS changes during infection and the mechanisms by which pathogens modulate AS remain poorly ***,we examined the global AS changes in tomato leaves infected with Phytophthora infestans,the infamous Irish famine *** show that more than 2000 genes exhibiting significant changes in AS are not differentially expressed,indicating that AS is a distinct layer of transcriptome reprogramming during plant-pathogen ***,our results show that *** subverts host immunity by repressing the AS of positive regulators of plant immunity and promoting the AS of susceptibility *** study the underlying mechanism,we established a luminescence-based AS reporter system in Nicotiana benthamiana to screen pathogen effectors modulating plant *** identified nine splicing regulatory effectors(SREs)from 87 *** *** studies revealed that SRE3 physically binds U1-70K to manipulate the plant AS machinery and subsequently modulates AS-mediated plant *** study not only unveils genome-wide plant AS reprogramming during infection but also establishes a novel AS screening tool to identify SREs from a wide range of plant pathogens,providing opportunities to understand the splicing regulatory mechanisms through which pathogens subvert plant immunity.

关键词： late blight Phytophthora infestans alternative splicing splicing regulatory effectors plant immunity tomato

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Genome-wide profiling of circular RNAs,alternative splicing,and R-loops in stem-differentiating xylem of Populus trichocarpa

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Journal of Integrative Plant Biology 2021年第7期63卷 1294-1308页

作者： Xuqing Liu Yubang Gao Jiakai Liao Miao Miao Kai Chen Feihu Xi Wentao Wei Huihui Wang Yongsheng Wang XiXu anireddy S.N.Reddy Lianfeng Gu Basic Forestry and Proteomics Research Center College of ForestryFujian Provincial Key Laboratory of Haixia Applied Plant Systems BiologyFujian Agriculture and Forestry UniversityFuzhou 350002China Basic Forestry and Proteomics Research Center College of Life ScienceFujian Agriculture and Forestry UniversityFuzhou 350002China Department of Biology and Program in Cell and Molecular Biology Colorado State UniversityFort CollinsColorado 80523USA

Circular RNAs(circRNAs)are a recently dis-covered type of non‐coding RNA derived from pre‐mRNAs.R‐loops consist of a DNA:RNA hybrid andthe associated single‐stranded ***-dopsis thaliana,circRNA:DNA R‐loops regulatealternative splicing(AS)ofSEPALLATA3(SEP3).However,the occurrence and functions ofcircRNAs and R‐loops inPopulus trichocarpaarelargely ***,we performed circRNA‐enriched sequencing in the stem‐differentiatingxylem(SDX)*** identified 2,742distinct circRNAs,including circ‐CESA4,circ‐IRX7,and circ‐GUX1,which are generated from genesinvolved in cellulose,and hemicellulose biosyn-thesis,*** investigate the roles ofcircRNAs in modulating alternative splicing(AS),we detected 7,836 AS events using PacBio Iso‐Seq and identified 634 circRNAs that overlappedwith 699 AS ***,using DNA:RNAhybrid immunoprecipitation followed by se-quencing(DRIP‐seq),we identified 8,932 R‐looppeaks that overlapped with 181 circRNAs and 672AS ***,several SDX‐related circRNAsoverlapped with R‐loop peaks,pointing to theirpossible roles in modulating AS in ***,overexpressing circ‐IRX7increased the levels ofR‐loop structures and decreased the frequency ofintron retention in *** provides a valuable R‐loop atlas resourceand uncovers the interplay between circRNAs andAS in SDX ***.

关键词： alternative splicing circular RNA R‐loop Populustrichocarpa stem‐differentiating xylem

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Ultra-Sensitive LC-MS/MS Method for the Trace Level Quantification of Six Potential Genotoxic Nitrosamine Impurities in Telmisartan

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American Journal of Analytical Chemistry 2021年第6期12卷 227-240页

作者： Kartheek Srinivas Chidella Vijaya Bharathi Dasari jayashree anireddy Center for Chemical Sciences and Technology Institute of Science and Technology Jawaharlal Nehru Technological University Hyderabad Hyderabad India Aurobindo Pharma Laboratory Research Center II Hyderabad India

Nitrosamine impurities are potentially genotoxic which are considered under cohort of concern as per ICH M7 guidelines and need to be controlled at trace levels during quantification in drug substances and drug products for safe human consumption. Recent regulatory requirements also suggest the need to have highly sensitive analytical methods for the accurate quantification of Nitrosamine impurities. In this paper we have presented simple, rapid and ultra-sensitive LC-MS/MS method for six potential genotoxic nitrosamine impurities: N-Nitroso dimethyl amine (NDMA), N-Nitroso diethyl amine (NDEA), N-Nitroso Ethyl Iso propylamine (NEIPA), N-Nitroso-N-methyl-4-aminobutyric acid (NMBA) N-Nitroso diisopropylamino (NDIPA) and N-Nitroso dibutyl amine (NDBA) with a LOQ of 0.004 ppm. Chromatographic separation is achieved using Zorbax SB C18 150 × 3.0 mm, 3.5 μ column with 0.1% formic acid in water as mobile phase A and 0.1% formic acid in methanol as mobile phase B at a flow rate of 0.3 ml/min using gradient mode of elution at a total run time of 18 minutes. Six nitrosamine impurities are successfully ionized and quantified in positive mode of atmospheric pressure chemical ionization (APCI) using multiple reaction monitoring (MRM). Method validation is performed as per ICH guidelines evaluating the limit of quantification and detection and found to give good S/N ratios with good linearity range of 0.002 - 2 ppm with regression coefficient >0.99 for all the six nitrosamine impurities. Method recoveries are established using three-step sample preparation protocol and are found to be satisfactory within 80% - 120%. The method can be used routinely applied for the detection of Nitrosamines in Telmisartan at a concentration of 1.5 ng/ml (0.03 ppm with respect to telmisartan concentration of 50 mg/ml).

关键词： Nitrosamine NDMA Genotoxic LC-MS/MS Telmisartan Impurities

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Simultaneous and Trace Level Quantification of Five Potential Genotoxic Impurities in Ranolazine Active Pharmaceutical Ingredient Using LC-MS/MS

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American Journal of Analytical Chemistry 2021年第1期12卷 1-14页

作者： Kartheek Srinivas Chidella Vijaya Bharathi Dasari jayashree anireddy Center for Chemical Sciences and Technology Institute of Science and Technology Jawaharlal Nehru Technological University Hyderabad Telangana India Aurobindo Pharma Laboratory Research Center II Hyderabad Telangana India

Highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed for the simultaneous determination of five potential genotoxic impurities in ranolazine active pharmaceutical ingredient. Chromatographic separation achieved using Poroshell C18 PFP 150 × 3.0 mm 2.7 μ column and 0.1% formic acid in water as Mobile phase A and 0.1% formic acid in methanol as mobile phase B using gradient elution and a flow rate of 0.4 ml/min with a run time of 18 minutes. Mass spectrometric conditions were optimized using electrospray ionization in positive mode. Method shows excellent linearity from 0.05 - 5.0 ppm of the ranolazine test concentration for all the five impurities. The correlation coefficient was observed greater than 0.99. Satisfactory recoveries were observed for all the five impurities within the range of 102.9% - 112.3%. Method has been validated as per ICH recommended guidelines with a LOQ of 0.15 ppm achieved. The developed method was able to quantify all the five impurities at a concentration level of 1 ng/ml (0.5 ppm with respect to 2 mg/ml ranolazine).

关键词： Genotoxic Impurities LC-MS/MS Sensitive

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Profiling of circular RNA N^(6)-methyladenosine in moso bamboo(Phyllostachys edulis)using nanopore-based direct RNA sequencing

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Journal of Integrative Plant Biology 2020年第12期62卷 1823-1838页

作者： Yongsheng Wang Huihui Wang Feihu Xi Huiyuan Wang Ximei Han Wentao Wei Hangxiao Zhang Qianyue Zhang Yushan Zheng Qiang Zhu Markus V.Kohnen anireddy S.N.Reddy Lianfeng Gu Basic Forestry and Proteomics Research Center College of life scienceFujian Agriculture and Forestry UniversityFuzhou 350002China Basic Forestry and Proteomics Research Center College of ForestryFujian Agriculture and Forestry UniversityFuzhou 350002China College of Forestry Fujian Agriculture and Forestry UniversityFuzhou 350002China Department of Biology and Program in Cell and Molecular Biology Colorado State UniversityFort CollinsColoradoUSA

N6-methyladenosine(m^(6)A)is a prevalent modification in messenger RNAs and circular RNAs that play important roles in regulating various aspects of RNA ***,the occurrence of the m^(6)A modification in plant circular RNAs has not been reported.A widely used method to identify m^(6)A modifications relies on m^(6)A-specific antibodies followed by next-generation sequencing of precipitated RNAs(MeRIP-Seq).However,one limitation of MeRIP-Seq is that it does not provide the precise location of m^(6)A at single-nucleotide *** more recent sequencing techniques such as Nanopore-based direct RNA sequencing(DRS)can overcome such limitations,the technology does not allow sequencing of circular RNAs,as these molecules lack a poly(A)***,we developed a novel method to detect the precise location of m^(6)A modifications in circular RNAs using Nanopore *** first enriched our samples for circular RNAs,which we then fragmented and sequenced on the Nanopore platform with a customized *** this method,we identified 470 unique circular RNAs from DRS reads based on the back-spliced junction *** exonic circular RNAs,about 10%contained m^(6)A sites,which mainly occurred around acceptor and donor splice *** study demonstrates the utility of our antibody-independent method in identifying total and methylated circular RNAs using Nanopore *** method has the additional advantage of providing the exact location of m^(6)A sites at single-base resolution in circular RNAs or linear transcripts from non-coding RNA without poly(A)tails.

关键词： circular precise overcome

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