ABSTRACT
Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.
Subject(s)
DNA, Complementary , High-Throughput Nucleotide Sequencing , Transglutaminases , Humans , Transglutaminases/genetics , Transglutaminases/metabolism , Substrate Specificity , DNA, Complementary/genetics , Amino Acid Sequence , Peptide LibraryABSTRACT
A novel, efficient and cost-effective approach for epitope identification of an antibody has been developed using a ribosome display platform. This platform, known as PURE ribosome display, utilizes an Escherichia coli-based reconstituted cell-free protein synthesis system (PURE system). It stabilizes the mRNA-ribosome-peptide complex via a ribosome-arrest peptide sequence. This system was complemented by next-generation sequencing (NGS) and an algorithm for analyzing binding epitopes. To showcase the effectiveness of this method, selection conditions were refined using the anti-PA tag monoclonal antibody with the PA tag peptide as a model. Subsequently, a random peptide library was constructed using 10 NNK triplet oligonucleotides via the PURE ribosome display. The resulting random peptide library-ribosome-mRNA complex was selected using a commercially available anti-HA (YPYDVPDYA) tag monoclonal antibody, followed by NGS and bioinformatic analysis. Our approach successfully identified the DVPDY sequence as an epitope within the hemagglutinin amino acid sequence, which was then experimentally validated. This platform provided a valuable tool for investigating continuous epitopes in antibodies.
Subject(s)
Peptide Library , Peptides , Epitope Mapping/methods , Cost-Benefit Analysis , Peptides/genetics , Peptides/chemistry , Antibodies, Monoclonal/genetics , Epitopes/genetics , Epitopes/chemistry , Ribosomes/genetics , High-Throughput Nucleotide Sequencing , Computational Biology , RNA, MessengerABSTRACT
BACKGROUND: The triglyceride-lowering drug, icosapent ethyl (IPE), was granted a new indication for the reduction of atherosclerotic cardiovascular disease risk in 2019. This study aimed to investigate the safety profile of IPE by mining the FDA Adverse Event Reporting System (FAERS) database. METHODS: The reporting odds ratio was used to analyze IPE's adverse events (AEs) based on the FAERS data from July 2012 to December 2022. We described the characteristics of AE reports and evaluated the clinical prioritization of AEs. Then we defined and analyzed nine interested adverse drug reactions (ADRs) in both overall and subgroups, and investigated the times to onset. RESULTS: The findings of our study strengthen the evidence for an increased risk of atrial fibrillation using IPE. IPE alone may not increase the risk of bleeding unless combined with antithrombotic drugs. Similar to statins, IPE alone can increase the risk of musculoskeletal pain, drug-related hepatic disorders, and hyperglycemia, but the risk could not double when IPE was combined with statins. Most ADRs occur in the early stage of treatment. CONCLUSIONS: This study provides a comprehensive real-world safety profile of IPE, which indicates that IPE is well-tolerated.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , Eicosapentaenoic Acid/analogs & derivatives , Hydroxymethylglutaryl-CoA Reductase Inhibitors , Humans , United States/epidemiology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/adverse effects , Adverse Drug Reaction Reporting Systems , Eicosapentaenoic Acid/adverse effects , United States Food and Drug AdministrationABSTRACT
cDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (1012) in a high throughput manner, based on their binding affinity. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions - 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position - 1 (Gln-Gln motif), and isoleucine or valine at position + 3. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules.
Subject(s)
GTP-Binding Proteins , Transglutaminases , Computational Biology , DNA, Complementary , GTP-Binding Proteins/metabolism , Glutamine/metabolism , High-Throughput Nucleotide Sequencing , Peptide Library , Peptides/chemistry , Protein Glutamine gamma Glutamyltransferase 2 , Substrate Specificity , Transglutaminases/metabolismABSTRACT
The medicinal plant Kadsura coccinea distributing in South China, was widely used for reducing inflammation and relieving pain. Previous study in our laboratory had proved the significant therapeutic effects of K. coccinea extract on adjuvant arthritis rats. To explore the responsible components and possible mechanisms, an AUF-HPLC-Q-TOF/ MS method was employed for screening and characterizing COX-2 ligands from K. coccinea stems for the first time. Meanwhile, the molecular docking was performed to simulate the binding modes for ligands and COX-2, the cell-free enzyme activity assay was applied to verify the direct COX-2 inhibition of potential inhibitors, and the cell-based study on COX-2 expression was to evaluate the anti-inflammatory effect of (+)-Anwulignan. As a result, the potential COX-2 inhibitor (+)-Anwulignan significantly suppressing COX-2 expressions in LPS signaling pathways might be a good candidate for anti-inflammation and analgesia. In conclusion, AUF mass spectrometry combining the molecular docking and bioassays in vitro was an efficient approach for discovering enzyme inhibitors from traditional herbs.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Cyclooxygenase 2 Inhibitors/pharmacology , Kadsura/chemistry , Animals , Anti-Inflammatory Agents/isolation & purification , China , Cyclooxygenase 2 Inhibitors/isolation & purification , Mice , Molecular Docking Simulation , Molecular Structure , Phytochemicals/isolation & purification , Phytochemicals/pharmacology , Plant Stems/chemistry , RAW 264.7 CellsABSTRACT
PM2.5-attributable deaths and years of life lost (YLL) due to specific causes during 1998-2015 in India were estimated using the integrated exposure-response (IER) model. The estimated PM2.5-mortality in India revealed an annual increasing rate of 2.7% during the study period. Spatially, deaths due to the exposure to ambient PM2.5 concentrated mostly in populated North India, and four northern states contributed 43% to the national PM2.5-attributable deaths in 2015. PM2.5-attributable deaths in India increased by 21% during 1998-2015 due to the changes of PM2.5 only, and deaths due to lung cancer (LC) revealed the largest sensitivity to increasing ambient PM2.5. The findings of this study suggest that aggressive air pollution control strategies should be implemented in North India due to their dominant contributions to the current health risks. Moreover, the rapid growth of LC related deaths with increasing ambient PM2.5 should not be neglected.
Subject(s)
Air Pollutants , Air Pollution , Lung Neoplasms , Air Pollutants/toxicity , Air Pollution/adverse effects , Air Pollution/analysis , Environmental Exposure/adverse effects , Humans , India/epidemiology , Mortality , Particulate Matter/analysis , Particulate Matter/toxicityABSTRACT
This study examines the sensitivity of daily PM2.5 to NOx emissions and meteorology using in situ observations from main cities of North China (NC). NC cities are divided into low-, medium-, and high-emission groups by the ranking of their 4-year mean NO2. For each emission group, daily NO2 levels are used to divide the days into good-, medium-, and bad-meteorological conditions. Regardless of their emission levels, all cities reveal significant decreases (96%-172%) in daily PM2.5 levels from bad to good meteorological conditions. The largest difference in PM2.5 concentrations between the emissions groups is found under bad meteorological conditions, with 56% higher PM2.5 in high-emission cities than low-emission cities, indicating PM2.5 under bad meteorological conditions has the largest sensitivity to emissions. The high-emission, bad-meteorology group saw a 24% decrease in mean daily PM2.5 levels from 2017, a high-emission year, to 2019, a low-emission year. However, under good meteorological conditions, the high-emissions group shows an increase of 8.8 µg/m3 in mean daily PM2.5 from 2017 to 2019 with a 2.6% increase in the possibility of high PM2.5. These results suggest the current emission reduction measures are more effective in controlling PM2.5 in high-emission cities under bad meteorological conditions than under other meteorological conditions.
ABSTRACT
Baeckea frutescens is a medicinal plant distributing from Southeast Asia to Australia. A pair of novel diastereomeric C-methylated triflavonoids named baeckeins L (1) and M (2) were isolated from the roots of B. frutescens. The structures of these isolates were elucidated by analysis of the 1D (1H/13C) and 2D NMR (HSQC/HMBC/NOESY) and HR-ESI-MS spectroscopic data, and the absolute configurations of chiral carbons (C-2/C-3/C-2°/C-3°) were established by CD spectrometry combined with quantum chemical calculations.
Subject(s)
Flavonoids/isolation & purification , Myrtaceae/chemistry , Plant Roots/chemistry , Plants, Medicinal/chemistry , Australia , Circular Dichroism , Flavonoids/chemistry , Molecular Conformation , Molecular Structure , Quantum Theory , Spectrum AnalysisABSTRACT
Analysis of observed PM2.5 in Beijing since 2009 reveals that winter haze over North China Plain (NCP) peaked in 2012 and 2013 and there was an improvement in air quality until 2016. The variation of wintertime PM2.5 from 2009 to 2016 is influenced by both emission changes and meteorology conditions, and we quantified the relative contributions from these two aspects. Sensitivity simulation by GEOS-Chem suggested that emission reductions over NCP in 2013-2017 caused 10% decrease of regional mean PM2.5 concentration in 2016 winter compared to 2012 winter level. We removed emission influence on PM2.5 to get PM2.5 that influenced by meteorology (met-influenced PM2.5). For met-influenced PM2.5, compared to original-observed PM2.5(the US Embassy data), percentage of clean days (daily PM2.5â¯≤â¯75⯵gâ¯m-3) decreases while that of polluted (75⯵gâ¯m-3â¯<â¯daily PM2.5 ≤150⯵gâ¯m-3) and heavily polluted (150⯵gâ¯m-3â¯<â¯daily PM2.5â¯≤â¯250⯵gâ¯m-3) days increases. However, proportion of extremely polluted (daily PM2.5â¯>â¯250⯵gâ¯m-3) days stays unchanged, even if emission reduction is doubled, indicating that the extremely polluted situation over NCP is dominated by meteorological conditions, and emission control from 2013 to 2017 has little effects on the extremely polluted days. We developed an effective haze day index (HDI) to represent the weather conditions conducive to haze days. HDI is constructed based on the normalized near surface meridional wind (V850), temperature difference (δT) between near surface (850â¯hPa) and upper atmosphere (250â¯hPa), and the relative humidity at 1000â¯hPa (RH1000). HDI is skillful to detect 72% of the severe haze days (daily PM2.5â¯>â¯150⯵gâ¯m-3). On average, the anomalously high V850 is the main cause of severe haze, while in 2012 winter, RH1000 favorable for secondary aerosols' formation is the largest contributor to haze.
Subject(s)
Aerosols/analysis , Air Pollutants/analysis , Air Pollution/analysis , Particulate Matter/analysis , Smog/analysis , Atmosphere/analysis , Beijing , China , Environmental Monitoring , Seasons , Weather , WindABSTRACT
This article marks the first report on high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) and quadruple time-of-flight mass spectrometry (Q-TOF/MS) for the identification and quantification of main bioactive constituents in Baeckea frutescens. In total, 24 compounds were identified or tentatively characterised based on their retention behaviours, UV profiles and MS fragment information. Furthermore, a validated method with good linearity, sensitivity, precision, stability, repeatability and accuracy was successfully applied for simultaneous determination of five flavonoids and one chromone in different plant parts of B. frutescens collected at different harvest times, and their dynamic contents revealed the appropriate harvest times. The established HPLC-DAD-Q-TOF/MS using multi-bioactive markers was proved to be a validated strategy for the quality evaluation on both raw materials and related products of B. frutescens.
Subject(s)
Chromatography, High Pressure Liquid/methods , Chromones/analysis , Flavonoids/analysis , Myrtaceae/chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Molecular Structure , Reproducibility of ResultsABSTRACT
Baeckea frutescens is an aromatic shrub used in South China as an ornamental and as a spice. Four unusual C-methylated biflavonoids named baeckeins F-I (1-4) were isolated from the roots of B. frutescens. The baeckeins F-I possessed a unique carbon skeleton, a flavonol conjugated with a coumaronochromone molecule via the unusual linkages of C-2-C-8 and C-3-O-C-7. Their structures were elucidated by analysis of the 1D ((1)H/(13)C) and 2D NMR (HSQC/HMBC/NOESY) and HR-ESI-MS spectroscopic data, and the absolute stereochemistry for chiral carbons of C-2 and C-3 was established by CD spectrometry combined with quantum chemical calculations. Baeckeins F-I (1-4) were also evaluated for their anti-inflammatory activities by detecting the NO production of LPS-induced RAW264.7 murine macrophage cell line; baeckein I (4) with the ß-d-glucose unit and configuration of (2R,3R) exhibited the highest NO inhibitory activity (IC50=15.2 µM), which was similar to that of the positive control indomethacin.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Biflavonoids/pharmacology , Drugs, Chinese Herbal/pharmacology , Myrtaceae/chemistry , Plant Roots/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/isolation & purification , Biflavonoids/chemistry , Biflavonoids/isolation & purification , Cell Line , Drugs, Chinese Herbal/chemistry , Drugs, Chinese Herbal/isolation & purification , Macrophages/drug effects , Macrophages/immunology , Mice , Molecular StructureABSTRACT
A phytochemical study of Baeckea frutescens afforded a new biflavonoid named as baeckein E (1), along with four known C-methylated flavonols, baeckeins A (2) and B (3), 6-methylquercetin (6) and 6-methylquercetin-4'-O-ß-D-glucopyranoside (7), and two known biflavonoids baeckeins C (4) and D (5). The structure of compound 1 was elucidated on the basis of spectroscopic analysis, including HR-ESI-MS, 1D and 2D NMR spectra, and the absolute configurations of C-2 and C-3 for compound 1 were assigned by circular dichroism spectrometry combined with quantum chemical calculations. The antioxidant and cytoprotective activities of compounds 1-7 were also investigated.
Subject(s)
Antioxidants/isolation & purification , Flavonoids/isolation & purification , Myrtaceae/chemistry , Flavonoids/chemistry , Molecular Structure , Plant Roots/chemistryABSTRACT
Baeckeins C (compound 1) and D (compound 2), a pair of new diastereomeric biflavonoids, were isolated from the roots of Baeckea frutescens. Their structures were elucidated on the basis of spectroscopic analysis, including HR-ESI-MS and one- and two-dimensional NMR spectra ((1)H and (13)C NMR, HSQC, HMBC, and ROESY). The absolute configurations of the 2,3-epoxide moiety for compounds 1 and 2 were determined by circular dichroism spectrometry combined with quantum chemical calculations.
Subject(s)
Flavonoids/isolation & purification , Myrtaceae/chemistry , Plant Roots/chemistry , Flavonoids/chemistry , Molecular Conformation , StereoisomerismABSTRACT
INTRODUCTION: The whole herb of Potentilla discolor has long been used for treatment of diarrhoea, malaria, haemoptysis and haematemesis in clinical applications. However, until now, there has been no literature regarding to the quality control of it. OBJECTIVE: To develop a simple and accurate HPLC-DAD (diode array detection)-ESI/MS(n) (electrospray ionisation multistage mass spectrometry) method for the identification of constituents in P. discolor and simultaneous quantification of its marker compounds. METHODOLOGY: Separations were performed on a Shimpack C18 column by gradient elution using acetonitrile:0.1% formic acid. The identification of constituents in P. discolor were achieved by HPLC-DAD-ESI/MS(n) while six flavonoids and five triterpenoids were determined by HPLC-DAD at 360 and 210 nm, repectively. RESULTS: A total of 23 compounds including 13 flavonoids and 10 triterpenoids were identified or tentatively characterised. A quantitative HPLC-DAD method allowing the simultaneously quantification of 11 marker compounds was optimised and validated for linearity, precision, accuracy, and limits of detection and quantification. The method was successfully applied to the quantification of 11 marker compounds in 10 samples of P. discolor collected from different provinces of China. CONCLUSION: This developed method was validated as simple, precise and accurate, and could be used for effectively and comprehensibly evaluating the quality of P. discolor.