Molecular authentication of commercial "Qian-hu" through the integration of nrDNA internal transcribed spacer 2 and nucleotide signature.

BACKGROUND: Peucedani Radix, also known as "Qian-hu" is a traditional Chinese medicine derived from Peucedanum praeruptorum Dunn. It is widely utilized for treating wind-heat colds and coughs accompanied by excessive phlegm. However, due to morphological similarities, limited resources, and heightened market demand, numerous substitutes and adulterants of Peucedani Radix have emerged within the herbal medicine market. Moreover, Peucedani Radix is typically dried and sliced for sale, rendering traditional identification methods challenging. MATERIALS AND METHODS: We initially examined and compared 104 commercial "Qian-hu" samples from various Chinese medicinal markets and 44 species representing genuine, adulterants or substitutes, utilizing the mini barcode ITS2 region to elucidate the botanical origins of the commercial "Qian-hu". The nucleotide signature specific to Peucedani Radix was subsequently developed by analyzing the polymorphic sites within the aligned ITS2 sequences. RESULTS: The results demonstrated a success rate of 100% and 93.3% for DNA extraction and PCR amplification, respectively. Forty-five samples were authentic "Qian-hu", while the remaining samples were all adulterants, originating from nine distinct species. Peucedani Radix, its substitutes, and adulterants were successfully identified based on the neighbor-joining tree. The 24-bp nucleotide signature (5'-ATTGTCGTACGAATCCTCGTCGTC-3') revealed distinct differences between Peucedani Radix and its common substitutes and adulterants. The newly designed specific primers (PR-F/PR-R) can amplify the nucleotide signature region from commercial samples and processed materials with severe DNA degradation. CONCLUSIONS: We advocate for the utilization of ITS2 and nucleotide signature for the rapid and precise identification of herbal medicines and their adulterants to regulate the Chinese herbal medicine industry.

Sequence confirmation of synthetic DNA exceeding 100 nucleotides using restriction enzyme mediated digestion combined with high-resolution tandem mass spectrometry.

Oligonucleotides have emerged as important therapeutic options for inherited diseases. In recent years, RNA therapeutics, especially mRNA, have been pushed to the market. Analytical methods for these molecules have been published extensively in the last few years. Notably, mass spectrometry has proven as a state-of-the-art quality control method. For RNA based therapeutics, numerous methods are available, while DNA therapeutics lack of suitable MS-based methods when it comes to molecules exceeding approximately 60 nucleotides. We present a method which combines the use of common restriction enzymes and short enzyme-directing oligonucleotides to generate DNA digestion products with the advantages of high-resolution tandem mass spectrometry. The instrumentation includes ion pair reverse phase chromatography coupled to a time-of-flight mass spectrometer with a collision induced dissociation (CID) for sequence analysis. Utilizing this approach, we increased the sequence coverage from 23.3% for a direct CID-MS/MS experiment of a 100 nucleotide DNA molecule to 100% sequence coverage using the restriction enzyme mediated approach presented in this work. This approach is suitable for research and development and quality control purposes in a regulated environment, which makes it a versatile tool for drug development.

Changes in the amount of nucleotide sugars in aged mouse tissues.

Aging affects tissue glycan profiles, which may alter cellular functions and increase the risk of age-related diseases. Glycans are biosynthesized by glycosyltransferases using the corresponding nucleotide sugar, and the availability of nucleotide sugars affects glycosylation efficiency. However, the effects of aging on nucleotide sugar profiles and contents are yet to be elucidated. Therefore, this study aimed to investigate the effects of aging on nucleotide sugars using a new LC-MS/MS method. Specifically, the new method was used to determine the nucleotide sugar contents of various tissues (brain, liver, heart, skeletal muscle, kidney, lung, and colon) of male C57BL/6NCr mice (7- or 26-month-old). Characteristic age-associated nucleotide sugar changes were observed in each tissue sample. Particularly, there was a significant decrease in UDP-glucuronic acid content in the kidney of aged mice and a decrease in the contents of several nucleotide sugars, including UDP-N-acetylgalactosamine, in the brain of aged mice. Additionally, there were variations in nucleotide sugar profiles among the tissues examined regardless of the age. The kidneys had the highest concentration of UDP-glucuronic acid among the seven tissues. In contrast, the skeletal muscle had the lowest concentration of total nucleotide sugars among the tissues; however, CMP-N-acetylneuraminic acid and CDP-ribitol were relatively enriched. Conclusively, these findings may contribute to the understanding of the roles of glycans in tissue aging.

A comprehensive evaluation of a polymeric zwitterionic hydrophilic monolith for nucleotide separation.

Rapid and effective separation of nucleotides (NTs) and their derivatives is crucial for studying their physiological functions. In this work, we comprehensively evaluated the separation ability of a zwitterionic hydrophilic monolith, i.e., poly(N,N-dimethyl-N-(3-methacrylamidopropyl)-N-(3-sulfopropyl)ammonium betaine-co-N,N'-methylenebisacrylamide) (poly(SPP-co-MBA)) for NTs analysis, including its selectivity, chemical stability under extremely basic condition and compatibility with hydrophilic interaction liquid chromatography (HILIC) coupled with mass spectrometry (HILIC-MS). The poly(SPP-co-MBA) monolith exhibited excellent chemical stability, as evidenced by the low relative standard deviation of retention time (0.16-1.05%) after 4000 consecutive injections over one month under strong alkaline elution condition (pH 10). After optimizing the separation conditions, including buffer pH and concentration, organic solvent content and column temperature, four nucleoside triphosphates, five nucleoside diphosphates and five nucleoside monophosphates were baseline separated within 7 min. Additionally, the mixtures containing one nucleoside and its corresponding mono-, di-, and triphosphates were baseline separated within only 3 min, respectively. It is good HILIC-MS compatibility was also confirmed by the satisfactory peak shape and high response of nine NTs. Overall, the proposed poly(SPP-co-MBA) monolith exhibited good mechanical stability and compatibility of HILIC-MS, making it a promising technique for NTs analysis.

Multiplexed LC-MS/MS quantification of salivary RNA modifications in periodontitis.

OBJECTIVE: To analyse the salivary epitranscriptomic profiles as periodontitis biomarkers using multiplexed mass spectrometry (MS). BACKGROUND: The field of epitranscriptomics, which relates to RNA chemical modifications, opens new perspectives in the discovery of diagnostic biomarkers, especially in periodontitis. Recently, the modified ribonucleoside N6-methyladenosine (m6A) was revealed as a crucial player in the etiopathogenesis of periodontitis. However, no epitranscriptomic biomarker has been identified in saliva to date. MATERIALS AND METHODS: Twenty-four saliva samples were collected from periodontitis patients (n = 16) and from control subjects (n = 8). Periodontitis patients were stratified according to stage and grade. Salivary nucleosides were directly extracted and, in parallel, salivary RNA was digested into its constituent nucleosides. Nucleoside samples were then quantified by multiplexed MS. RESULTS: Twenty-seven free nucleosides were detected and an overlapping set of 12 nucleotides were detected in digested RNA. Among the free nucleosides, cytidine and three other modified nucleosides (inosine, queuosine and m6Am) were significantly altered in periodontitis patients. In digested RNA, only uridine was significantly higher in periodontitis patients. Importantly there was no correlation between free salivary nucleoside levels and the levels of those same nucleotides in digested salivary RNA, except for cytidine, m5C and uridine. This statement implies that the two detection methods are complementary. CONCLUSION: The high specificity and sensitivity of MS allowed the detection and quantification of multiple nucleosides from RNA and free nucleosides in saliva. Some ribonucleosides appear to be promising biomarkers of periodontitis. Our analytic pipeline opens new perspectives for diagnostic periodontitis biomarkers.

Development of an Artificially Intelligent Nanopore for High-Throughput DNA Sequencing with a Machine-Learning-Aided Quantum-Tunneling Approach.

Solid-state nanopore-based single-molecule DNA sequencing with quantum tunneling technology poses formidable challenges to achieve long-read sequencing and high-throughput analysis. Here, we propose a method for developing an artificially intelligent (AI) nanopore that does not require extraction of the signature transmission function for each nucleotide of the whole DNA strand by integrating supervised machine learning (ML) and transverse quantum transport technology with a graphene nanopore. The optimized ML model can predict the transmission function of all other nucleotides after training with data sets of all the orientations of any nucleotide inside the nanopore with a root-mean-square error (RMSE) of as low as 0.062. Further, up to 96.01% accuracy is achieved in classifying the unlabeled nucleotides with their transmission readouts. We envision that an AI nanopore can alleviate the experimental challenges of the quantum-tunneling method and pave the way for rapid and high-precision DNA sequencing by predicting their signature transmission functions.

Beyond the GTP-cap: Elucidating the molecular mechanisms of microtubule catastrophe.

Almost 40 years since the discovery of microtubule dynamic instability, the molecular mechanisms underlying microtubule dynamics remain an area of intense research interest. The "standard model" of microtubule dynamics implicates a "cap" of GTP-bound tubulin dimers at the growing microtubule end as the main determinant of microtubule stability. Loss of the GTP-cap leads to microtubule "catastrophe," a switch-like transition from microtubule growth to shrinkage. However, recent studies, using biochemical in vitro reconstitution, cryo-EM, and computational modeling approaches, challenge the simple GTP-cap model. Instead, a new perspective on the mechanisms of microtubule dynamics is emerging. In this view, highly dynamic transitions between different structural conformations of the growing microtubule end - which may or may not be directly linked to the nucleotide content at the microtubule end - ultimately drive microtubule catastrophe.

rRNA expansion segment 7 in eukaryotes: from Signature Fold to tentacles.

The ribosomal core is universally conserved across the tree of life. However, eukaryotic ribosomes contain diverse rRNA expansion segments (ESs) on their surfaces. Sites of ES insertions are predicted from sites of insertion of micro-ESs in archaea. Expansion segment 7 (ES7) is one of the most diverse regions of the ribosome, emanating from a short stem loop and ranging to over 750 nucleotides in mammals. We present secondary and full-atom 3D structures of ES7 from species spanning eukaryotic diversity. Our results are based on experimental 3D structures, the accretion model of ribosomal evolution, phylogenetic relationships, multiple sequence alignments, RNA folding algorithms and 3D modeling by RNAComposer. ES7 contains a distinct motif, the 'ES7 Signature Fold', which is generally invariant in 2D topology and 3D structure in all eukaryotic ribosomes. We establish a model in which ES7 developed over evolution through a series of elementary and recursive growth events. The data are sufficient to support an atomic-level accretion path for rRNA growth. The non-monophyletic distribution of some ES7 features across the phylogeny suggests acquisition via convergent processes. And finally, illustrating the power of our approach, we constructed the 2D and 3D structure of the entire LSU rRNA of Mus musculus.

Active compound analysis of Ziziphus jujuba cv. Jinsixiaozao in different developmental stages using metabolomic and transcriptomic approaches.

Jujube (Ziziphus jujuba Mill.) is a popular fruit with health benefits ascribed to its various metabolites. These metabolites determine the flavors and bioactivities of the fruit, as well as their desirability. However, the dynamics of the metabolite composition and the underlying gene expression that modulate the overall flavor and accumulation of active ingredients during fruit development remain largely unknown. Therefore, we conducted an integrated metabolomic and transcriptomic investigation covering various developmental stages in the jujube cultivar Z. jujuba cv. Jinsixiaozao, which is famous for its nutritional and bioactive properties. A total of 407 metabolites were detected by non-targeted metabolomics. Metabolite accumulation during different jujube developmental stages was examined. Most nucleotides and amino acids and their derivatives accumulated during development, with cAMP increasing notably during ripening. Triterpenes gradually accumulated and were maintained at high concentrations during ripening. Many flavonoids were maintained at relatively high levels in early development, but then rapidly decreased later. Transcriptomic and metabolomic analyses revealed that chalcone synthase (CHS), chalcone isomerase (CHI), flavonol synthase (FLS), and dihydroflavonol 4-reductase (DFR) were mainly responsible for regulating the accumulation of flavonoids. Therefore, the extensive downregulation of these genes was probably responsible for the decreases in flavonoid content during fruit ripening. This study provide an overview of changes of active components in 'Jinsixiaozao' during development and ripening. These findings enhance our understanding of flavor formation and will facilitate jujube breeding for improving both nutrition and function.

Effect of different drying methods on free amino acid and flavor nucleotides of scallop (patinopecten yessoensis) adductor muscle.

The effects of hot air drying (HAD), vacuum hot air drying (VHAD), microwave drying (MWD), and vacuum freeze drying (VFD) on free amino acids (FAAs) and flavor nucleotides in scallop adductor muscle (SAM) were studied. The liquid chromatography and multidimensional infrared spectroscopy (MM-IR) were used. Compared with fresh SAM, the main FAAs were glycine, alanine, arginine, and glutamic acid in dried SAM. The total FAAs content in VFD group was 1.40-1.90 times of the other group. The umami taste nucleotides (IMP and AMP) content in the VFD and MWD groups was significantly higher than that in HAD and VHAD groups. Equivalent umami concentrations were found: VFD > MWD > VHAD > HAD. MM-IR analysis was an efficient method for identifying taste components. The results revealed FAAs and flavor nucleotides and the mutual adjustment of compounds were related to drying method, and VFD was preferred for taste substance retention in scallops.

Study on nucleotide, myofibrillar protein biochemical properties and microstructure of freeze-dried scallop striated muscle during storage and rehydration.

The biochemical properties and microstructural changes of freeze-dried Japanese scallop (Patinopecten yessoensis) striated muscle during room temperature storage and rehydration were investigated. The results showed that the content of ATP in freeze-dried scallop muscle remained stable with no significant difference (p > 0.05). However, ATP was rapidly decomposed and AMP accumulated within 1.5 min of rehydration, and HxR and Hx were gradually produced from AMP decomposition with the extension of rehydration time. Besides, the results of chymotryptic digestion patterns demonstrated that the rod of myosin was unstable after dehydration, reflecting lower salt solubility than that of frozen-thawed scallop. In contrast, the myosin subfragment-1 (S-1) was stable, as indicated by the constant of Ca2+-ATPase activity of freeze-dried scallops throughout the storage and rehydration (p > 0.05). Furthermore, the microstructural analysis revealed that the Z line of the freeze-dried scallop was broken after dehydration process. This study might be useful for developing high-quality dehydrated scallops in the future.

Metabolomic profiling of Dezhou donkey seminal plasma related to freezability.

Donkeys are indispensable livestock in China because they have transport function and medicinal value. With the popularization of artificial insemination on donkeys, semen cryopreservation technology has gradually become a research hotspot. Seminal plasma is a necessary medium for transporting sperm and provides energy and nutrition for sperm. Seminal plasma metabolites play an important role in the process of sperm freezing, and also have an important impact on sperm motility and fertilization rate after freezing and thawing. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to compare the metabolic characteristics of seminal plasma of high freezability (HF) and low freezability (LF) male donkeys. We identified 672 metabolites from donkey seminal plasma, of which 33 metabolites were significantly different between the two groups. Metabolites were identified and categorized according to their major chemical classes, including homogeneous non-metal compounds, nucleosides, nucleotides, and analogues, organosulphur compounds, phenylpropanoids and polyketide, organoheterocyclic compounds, organic oxygen compounds, benzenoids, organic acids and derivatives, lipids and lipid-like molecules, organooxygen compounds, alkaloids and derivatives, organic nitrogen compounds. The results showed that the contents of phosphatidylcholine, piceatannol and enkephalin in donkey semen of HF group were significantly higher than those of LF group (p < .05), while the contents of taurocholic and lysophosphatidic acid were significantly lower than those of LF group (p < .05). The different metabolites were mainly related to sperm biological pathway response and oxidative stress. These metabolites may be considered as candidate biomarkers for different fertility in jacks.

Purification and Analysis of Nucleotides and Nucleosides from Plants.

This protocol describes necessary steps to isolate and quantify nucleotides and nucleosides from plant samples. Proper sample preparation in combination with liquid chromatography coupled to mass spectrometry enables the sensitive detection and quantification of metabolites of low abundance. Utilizing a liquid-liquid extraction in combination with a weak anion-exchange solid phase extraction enables the separation of negatively charged molecules from uncharged metabolites or cations.

Signalling by extracellular nucleotides in health and disease.

Nucleotides are released from all cells through regulated pathways or as a result of plasma membrane damage or cell death. Outside the cell, nucleotides act as signalling molecules triggering multiple responses via specific plasma membrane receptors of the P2 family. In the nervous system, purinergic signalling has a key function in neurotransmission. Outside the nervous system, purinergic signalling is one of the major modulators of basal tissue homeostasis, while its dysregulation contributes to the pathogenesis of various disease, including inflammation and cancer. Pre-clinical and clinical evidence shows that selective P2 agonists or antagonists are effective treatments for many pathologies, thus highlighting the relevance of extracellular nucleotides and P2 receptors as therapeutic targets.

The CAR-mRNA Interaction Surface Is a Zipper Extension of the Ribosome A Site.

The ribosome CAR interaction surface behaves as an extension of the decoding center A site and has H-bond interactions with the +1 codon, which is next in line to enter the A site. Through molecular dynamic simulations, we investigated the codon sequence specificity of this CAR-mRNA interaction and discovered a strong preference for GCN codons, suggesting that there may be a sequence-dependent layer of translational regulation dependent on the CAR interaction surface. Dissection of the CAR-mRNA interaction through nucleotide substitution experiments showed that the first nucleotide of the +1 codon dominates over the second nucleotide position, consistent with an energetically favorable zipper-like activity that emanates from the A site through the CAR-mRNA interface. Moreover, the CAR/+1 codon interaction is affected by the identity of nucleotide 3 of +1 GCN codons, which influences the stacking of G and C. Clustering analysis suggests that the A-site decoding center adopts different neighborhood substates that depend on the identity of the +1 codon.

An Overview of Pretreatment and Analysis of Nucleotides in Different Samples (Update since 2010).

Nucleotides, which are important low-molecular-weight compounds present in organisms, are precursors of nucleic acids and participate in various regulatory and metabolic functions. Sensitive and valid methods for monitoring and determining nucleotides and nucleosides in different samples are urgently required. Due to the presence of numerous endogenous interferences in complex matrices and the high polarity of the molecules of the phosphate moiety, the determination of nucleotide content is challenging. This review summarizes the pretreatment and analysis methods of nucleotides in different samples. Advanced pretreatment methods, including different microextraction methods, solid-phase extraction based on novel materials, QuEChERS, are clearly displayed, and continuous progress which has been made in LC, LC-MS/MS and capillary electrophoresis methods are discussed. Moreover, the strengths and weaknesses of different methods are discussed and compared. Highlight:Advanced pretreatment and detection methods of nucleotides were critically reviewed.Microextraction technology was one of the trends of nucleotides pretreatment in the future.Applications of novel materials and supercritical fluid were highlighted.The evolution and advance of HRMS analyzers were in detailed.

Taste compounds generation and variation of broth in pork meat braised processing by chemical analysis and an electronic tongue system.

The aim was to unveil the generation and variation rule of the main taste components in braised broth for 10 quantitative repeated braising cycles. The major taste compounds of three groups (MS, broth cooked with meat and spices; M, broth cooked with meat; and S, broth cooked with spices) were systematically analyzed by the state-of-art chromatography and electronic sensory technology. As braising cycles progressed, contents of free 5'-nucleotides and amino acids were increased in MS and M, while those nucleotides were not detected in S. A significant discrimination of taste in MS and M was revealed by electronic tongue evaluation during the process. As the formation rates (FR) of taste compounds and the transformation rates (TR) of taste compounds to volatile compounds were mainly accounting for the generation and variation of flavor in broth, a hypothesis was proposed to illustrate the whole variation of taste compounds in the process integrally that the ratio of FR/TR dividing the process into three stages, Degradation, Balance, and Accumulation. PRACTICAL APPLICATIONS: The traditional braising process and formula are empirical and extensive, which impede the increase in meat products output. Nowadays, the industry of braising products is facing a problem of standardization and quality control, and needs to carry out scientific and quantitative process improvement efficiently. Therefore, the developed comprehensive approach demonstrates great potential for braised meat broth flavor monitoring and quality control in an objective and holistic manner. It provides data support and new ideas of technology development for quality control in the process of meat braising.

Analytical characterization of nucleotides and their concentration variation in drinking water treatment process.

Nucleotides, as the basic building blocks of nucleic acids, widely exist in aqueous environment. In this study, we developed a solid phase extraction-high performance liquid chromatography-tandem mass spectrometry (SPE-UPLC-MS/MS) method for the analysis of 5'-adenosine monophosphate (AMP), 5'-uridine monophosphate (UMP), 5'-cytidine monophosphate (CMP) and 5'-guanosine monophosphate (GMP). The method achieved limits of detection (LODs) of 0.1-1.0 ng/L, and recoveries of 85-95% for the four tested nucleotides. The occurrence and concentrations of the four nucleotides in water from eight representative drinking water treatment and distribution systems in China were determined using this method. All four nucleotides were detectable in water treatment plant (WTP) influent and effluent, at concentrations of up to 30 ng/L and with occurrence frequency of around 90%. The concentrations of identified nucleotides increased 3-10 times after 10 km of water age in the water distribution system. Biological filters and coagulation increased the concentrations of nucleotides, conversely, active carbon, ozonation, and ultrafiltration membrane removed nucleotides in water. The effects of active carbon and coagulation were further confirmed using laboratory-controlled experiment. In addition, monochlorinated nucleotides were identified as the chlorination products of nucleotides.

Versatile separation of nucleotides from bacterial cell lysates using strong anion exchange chromatography.

Nucleotides exemplify some of the building blocks of life, comprising DNA & RNA, participating in processes such as cell signaling and metabolism, and serving as carriers of metabolic energy. The quantification of these compounds in biological samples is critical for researchers to understand complex systems. Herein, we demonstrate an anion exchange chromatography method utilizing a pH range of 8 to 10, which provides superior resolution and selectivity to previously reported methods and, more importantly, gives the flexibility to shift analyte selectivity if resolution between analytes is not optimal. We have applied the method to study the kinetics of the nucleotide pool in a bacterial cell-free lysate system that is producing RNA. Sample to sample runtimes are less than 18 min and recoveries greater than 96% were observed for all analytes through our methanol quench protocol with day-to-day variabilities less than 5%. This method reliably detects and quantifies all analytes that were expected to be observed in the process and helps lay the groundwork for future nucleotide research.

DNA bridges: A novel platform for single-molecule sequencing and other DNA-protein interaction applications.

DNA molecular combing is a technique that stretches thousands of long individual DNA molecules (up to 10 Mbp) into a parallel configuration on surface. It has previously been proposed to sequence these molecules by synthesis. However, this approach poses two critical challenges: 1-Combed DNA molecules are overstretched and therefore a nonoptimal substrate for polymerase extension. 2-The combing surface sterically impedes full enzymatic access to the DNA backbone. Here, we introduce a novel approach that attaches thousands of molecules to a removable surface, with a tunable stretching factor. Next, we dissolve portions of the surface, leaving the DNA molecules suspended as 'bridges'. We demonstrate that the suspended molecules are enzymatically accessible, and we have used an enzyme to incorporate labeled nucleotides, as predicted by the specific molecular sequence. Our results suggest that this novel platform is a promising candidate to achieve high-throughput sequencing of Mbp-long molecules, which could have additional genomic applications, such as the study of other protein-DNA interactions.