WITHDRAWN: Chloroplast genome evolution of Berberis (Berberidaceae): Implications for phylogeny and metabarcoding.

After consultation with external experts, the authors acknowledged discrepancies in the classification of certain Berberis samples discussed in the article. Berberis is one of the most complex plant genera, and identifications are very hard, limited to only a handful of experts due to rampant hybridizations and other issues of reticulate evolution. This article has therefore been withdrawn at the request of the authors and with the consent of the editor until the species identification issue has been resolved. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Qualitative identification of lonicerae japonicae flos in traditional chinese medicine using metabarcoding combined with specific mini-barcodes.

BACKGROUND: Lonicerae japonicae flos, also known as Jinyinhua (JYH), is an important component of traditional Chinese patent medicine (TCPM) products. However, the potential for adulteration and substitution with low-quality materials highlights the need for a reliable and sensitive approach to identify the species composition of TCPM products for consumer safety. METHODS AND RESULTS: We used universal ITS2 primers to amplify TCPMs containing JYH. However, the results were inconclusive, as only one operational taxonomic unit (OTU) was identified as Lonicera sp., which could not be identified at the species level. To confirm the species identification of Lonicera sp. in TCPM, we developed a short mini-barcode primer based on the psbA-trnH region, which, in combination with DNA metabarcoding technology, allowed for qualitative and quantitative analysis of artificially mixed samples. We applied the mini-barcode to distinguish TCPMs containing JYH and demonstrated its relatively accurate quantitative ability in identifying two Lonicera species. CONCLUSIONS: Our study presents a method for qualitative and quantitative identification of JYH, providing a promising application of DNA metabarcoding technology in the quality control of TCPM products.

Species diversity analysis of commercial Mantidis Ootheca samples contaminated by store pests based on DNA metabarcoding.

Mantidis Ootheca (Sangpiaoxiao, mantis egg case) is a typical multi-origin Chinese medicinal material. The Chinese Pharmacopoeia stipulates that the Mantidis Ootheca originates from three species of Mantis: Tenodera sinensis, Statilia maculate, and Hierodula patellifera. However, Mantidis Ootheca mainly relies on field collection, which leads to confusion of its actual origin in the market. As the clinical use of Mantidis Ootheca with unknown original mantis species will pose potential risks to drug safety, it is necessary to survey the commercially available Mantidis Ootheca origin species. However, as the egg case of Mantis, the morphological characters of Mantidis Ootheca are limited and usually cannot serve as accurate identification tool. DNA barcoding, which is widely used in taxonomic studies of animals, is severely affected by the impact of storage pests and DNA degradation. Thus, this study collected a total of 4580 Mantidis Ootheca and pooled separately Mantidis Ootheca samples according to 18 different sources as DNA samples to analyze the origin diversity of Mantidis Ootheca individuals contaminated by common store pests collected in in the market using DNA metabarcoding, and to provide a basis for quality control of Mantidis Ootheca. 37 Mantis ASVs and 9 Mantis MOTUs were identified through species delimitation, and the high-level intraspecific diversity was depicted as haplotype network plot. Besides Tenodera sinensis and Hierodula patellifera as genuine original mantis species defined in the Chinese Pharmacopoeia, Tenodera angustipennis was also the origin species of these Mantidis Ootheca samples.

Transcriptome and complete chloroplast genome of Glycyrrhiza inflata and comparative analyses with the other two licorice species.

In this study, we characterized the transcriptome and chloroplast genome of Glycyrrhiza inflata and performed comparative analyses with G. uralensis and G. glabra. 60,541unigenes were obtained from the transcriptome of G. inflata. The results of function annotation revealed a similar distribution of functional categories among three licorice species. By comparing chloroplast genomes of licorice species, it was demonstrated that the structure and the length of genome as well as gene content and gene order were highly similar. The phylogenetic tree, constructed with the mixed data of transcriptome and chloroplast genome, elucidated that G. inflata and G. glabra had a closer relationship than G. uralensis. Six regions were suggested as potential markers for the identification of three licorice species. In each licorice species, two unigenes were homologous to reference flavonol synthase. For G. inflata, 48 and 21 RNA editing sites were detected by PREP-Cp program and RNA-Seq data mapping, respectively.

Cloning and Characterization of Two Iridoid Synthase Homologs from Swertia Mussotii.

Swertia mussotii is an important medicinal plant found on the Qinghai Tibetan Plateau that has great economic and medicinal value. This plant has enjoyed a long history of use as a curative for hepatitis. The biological activity of secoiridoids, including gentiopicroside and swertiamarin, has been mainly tested for its anti-hepatitis effects. Here, we identify two candidate genes (SmIS1 and SmIS2) that are homologues of iridoid synthase and that are components of the secoiridoid pathway in S. mussotii. Using sequencing and phylogenetic analyses, we confirm that SmIS1 and SmIS2 contain six conserved short-chain dehydrogenases/reductase (SDR) motifs and thus belong to the P5ßRs group. The two purified Escherichia coli-expressed proteins reduced 8-oxogeranial to both nepetalactol and iridodials. A comparison of the kinetic parameters of SmIS1 and SmIS2 recombinant proteins revealed that SmIS2 has a lower affinity than SmIS1 for 8-oxogeranial. Transcript levels of the two genes were analysed in three different tissues of S. mussotii using semi-quantitative RT-PCR and RT-qPCR. SmIS1 and SmIS2 expression levels were more abundant in leaves and stems. This investigation adds to our knowledge of P5ßRs genes in the secoiridoid synthesis pathway and provides candidate genes for genetically improving S. mussotii by enhancing secondary metabolite production.

PacBio full-length transcriptome profiling of insect mitochondrial gene expression.

In this study, we sequenced the first full-length insect transcriptome using the Erthesina fullo Thunberg based on the PacBio platform. We constructed the first quantitative transcription map of animal mitochondrial genomes and built a straightforward and concise methodology to investigate mitochondrial gene transcription, RNA processing, mRNA maturation and several other related topics. Most of the results were consistent with the previous studies, while to the best of our knowledge some findings were reported for the first time in this study. The new findings included the high levels of mitochondrial gene expression, the 3' polyadenylation and possible 5' m(7)G caps of rRNAs, the isoform diversity of 12S rRNA, the polycistronic transcripts and natural antisense transcripts of mitochondrial genes et al. These findings could challenge and enrich fundamental concepts of mitochondrial gene transcription and RNA processing, particularly of the rRNA primary (sequence) structure. The methodology constructed in this study can also be used to study gene expression or RNA processing of nuclear genomes.

Delineation of Platelet Activation Pathway of Scutellarein Revealed Its Intracellular Target as Protein Kinase C.

Erigeron breviscapus has been widely used in traditional Chinese medicine (TCM) and its total flavonoid component is commonly used to treat ischemic stroke, coronary heart disease, diabetes and hypertension. Scutellarin is the major ingredient of E. breviscapus and scutellarein is one of the main bioactive metabolites of scutellarin in vivo, but the latter's pharmacological activities have not been fully characterized. Provided evidence that could inhibit platelet aggregation, the effect of scutellarein on rat washed platelets and its underlying mechanisms were evaluated in our research. Scutellarein inhibited platelet adhesion and aggregation induced by multiple G protein coupled receptor agonists such as thrombin, U46619 and ADP, in a concentration-dependent manner. Furthermore, the mild effect of scutellarein on intracellular Ca(2+) mobilization and cyclic AMP (cAMP) level was observed. On the other hand, the role of scutellarein as potential protein kinase C (PKC) inhibitor was confirmed by PKC activity analysis and molecular docking. The phorbol myristate acetate-induced platelets aggregation assay with or without ADP implied that the scutellarein takes PKC(s) as its primary target(s), and acts on it in a reversible way. Finally, scutellarein as a promising agent exhibited a high inhibition effect on ADP-induced platelet aggregation among its analogues. This study clarifies the PKC-related signaling pathway involved in antiplatelet action of scutellarein, and may be beneficial for the treatment of cardiovascular diseases.

The Complete Chloroplast Genome Sequence of the Medicinal Plant Swertia mussotii Using the PacBio RS II Platform.

Swertia mussotii is an important medicinal plant that has great economic and medicinal value and is found on the Qinghai Tibetan Plateau. The complete chloroplast (cp) genome of S. mussotii is 153,431 bp in size, with a pair of inverted repeat (IR) regions of 25,761 bp each that separate an large single-copy (LSC) region of 83,567 bp and an a small single-copy (SSC) region of 18,342 bp. The S. mussotii cp genome encodes 84 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. The identity, number, and GC content of S. mussotii cp genes were similar to those in the genomes of other Gentianales species. Via analysis of the repeat structure, 11 forward repeats, eight palindromic repeats, and one reverse repeat were detected in the S. mussotii cp genome. There are 45 SSRs in the S. mussotii cp genome, the majority of which are mononucleotides found in all other Gentianales species. An entire cp genome comparison study of S. mussotii and two other species in Gentianaceae was conducted. The complete cp genome sequence provides intragenic information for the cp genetic engineering of this medicinal plant.

[Interaction of anti-thrombotic and anti-inflammatory activities of commonly used traditional Chinese medicine for promoting blood circulation and removing blood stasis revealed by network pharmacology analysis].

Chinese traditional patent medicine for promoting blood circulation and removing blood stasis(PBCRBS) originated from traditional Chinese medicine theory and had approved efficacy and safety standards. However, its compatibility regularity and anti-thrombotic mechanism is not clear. To analyze the compatibility regularity and anti-thrombotic mechanism of Chinese traditional patent medicine for PBCRBS, a statistical and bioinformatics analysis was carried out using traditional Chinese medicine inheritance support system (TICMISS, V2.0) and ingenuity pathway analysis (IPA). The compatibility regularity analysis shows that the most commonly used herb combinations are Danshen (Salvia miltiorrhiza Bge.), Chuanxiong (Ligusticum chuanxiong Hort.) and Honghua (Carthamustinctorius L.). The anti-thrombotic mechanism analysis reveals that 25 ingredients have an effect on 29 thrombosis related molecules which 23 molecules are related to inflammation response. Furthermore, there are 5 inflammation molecules (NOS2, PTGS2, IL6, TNF, IL1ß) served as major targets. At the same time, Danshen, Chuangxiong and Honghua mainly used as sovereign herb or minister herb in the application of cardiovascular and cerebrovascular diseases. Therefore, Chinese traditional patent medicine for PBCRBS probably has an effect on anti-thrombotic activity through inhibiting the inflammatory response. In summary, the most commonly used herb combinations of Chinese traditional patent medicine for PBCRBS are Danshen, Chuanxiong and Honghua. Inhibiting inflammatory response, especially inflammation related molecules (NOS2, PTGS2, IL6, TNF and IL1ß), is probably a new starting point to clarify the anti-thrombotic mechanism of Chinese patent medicine for PBCRBS.

A Prognostic Risk Signature of Two Autophagy-Related Genes for Predicting Triple-Negative Breast Cancer Outcomes.

Background: Triple-negative breast cancer (TNBC) is recognized as the most aggressive molecular subtype of breast cancer. Recent studies have highlighted the complex role of autophagy in the pathogenesis of TNBC. Methods: In this study, we evaluated 18,330 genes, including 1111 autophagy-related genes, (ARGs), across 579 TNBC samples from online databases. Differentially expressed ARGs in TNBC were identified using high-throughput RNA-seq data from the Cancer Genome Atlas (TCGA). Prognostic factors were examined through Cox regression and multivariate Cox analyses, with predictive efficacy assessed using receiver operating characteristic (ROC) curves. A nomogram integrating the risk signature with clinicopathological factors, such as TNM stage, was developed. Immunohistochemical analysis of clinical samples was also conducted. Results: EIF4EBP1 and NPAS3 were significantly correlated with prognostic outcomes in patients with TNBC. Multivariate Cox regression analysis demonstrated that the expression levels of these two genes were accurate predictors of disease progression in TNBC samples from TCGA and the GSE31519 dataset. The efficacy of this predictive model was validated using ROC curve analysis and calibration plots, confirming its ability to accurately estimate the 1-, 2-, and 3-year survival rates for individuals with TNBC. Additionally, EIF4EBP1 and NPAS3 expression influenced drug sensitivity in TNBC cell lines, with notably lower NPAS3 expression in TNBC tissues, particularly in Stage III cases. This study is the first to report NPAS3 expression in patients with TNBC. Conclusion: The autophagy-related genes EIF4EBP1 and NPAS3 may serve as independent prognostic factors for individuals with TNBC.

The metabarcoding of Grubs: Traditional herbal medicine of Scarabaeidae larvae.

Grubs, called Qicao in China, have a long tradition as herbal medicine in East Asia. These larvae belong to the diverse family Scarabaeidae and are typically harvested from the wild during their immature stage based on morphological characteristics. However, rapid and accurate identification becomes challenging when relying solely on external morphological features, as the lack of clarity on biological sources raises safety concerns for clinical applications. The application of DNA metabarcoding provides a solution by enabling the determination of the biological source of a large sample. In the current study, we collected 19 batches of Grubs, consisting of 11,539 individuals, from the market and analyzed their biological composition through metabarcoding. We identified 49 Amplicon Sequence Variants (ASVs), 21 of which were Grubs. The 21 ASVs were classified into seven Molecular Operational Taxonomic Units (MOTUs) through species delimitation, which revealed that commercially available Grubs are predominantly sourced from Protaetia brevitarsis seulensis, while species of Rutelinae, Anomala, and Holotrichia were also abundant in some commercial batches. Among the identified ASVs, 28 belonged to non-Grub species and indicated adulteration from different animal families; high abundances of these ASVs were detected for Bombycidae, Tabanidae, and Viviparidae. Our findings underscore the complexity of Grubs' species composition and advocate for a deeper understanding of the wildlife sources contributing to herbal products. This research contributes valuable insights into the molecular identification of Grubs, paving the way for enhanced quality assurance in traditional medicine applications to provide safe and effective medicines for humanity.

Exploring the biological diversity and source species of medicinal horseflies through metabarcoding.

Horseflies from the Tabanidae family play a significant role in traditional Chinese medicine to treat various health conditions, including coronary heart disease, stroke, headaches, liver cirrhosis, psoriasis, and hepatic carcinoma. There are 27 species of Tabaninae (Tabanidae) used as medicine, and they showed high morphological similarities with those for which medicinal properties have not been reported. Nonetheless, there have been reports suggesting that medicinal crude drugs sometimes contain irrelevant or false species, impacting the drug's efficacy. In this current study, we collected 14 batches, totaling 13,528 individuals, from various provinces in China. Instead of "classic" DNA barcoding strategy, we employed a high-throughput metabarcoding approach to assess the biological composition of crude drug mixtures derived from horseflies. Our analysis identified 40 Amplicon Sequence Variants (ASVs) with similarity percentages ranging from 92% to 100% with 12 previously reported species. Species delimitation methods revealed the presence of 11 Molecular Operational Taxonomic Units (MOTUs), with ten belonging to the Tabanus genus and one to Hybomitra. Tabanus sp6 displayed the highest relative abundance, and its ASVs showed close resemblance to Tabanus pleski. Our investigations revealed that the medicinal batches were biologically composed of 6 to 12 species. Some batches contained ASVs that closely resembled species previously associated with false Tabanus species. In conclusion, our findings offer valuable insights into the biological composition of crude drugs derived from horseflies and have the potential to enhance the quality of these traditional medicines.

Corrigendum: Development of mini-barcode based on chloroplast genome and its application in metabarcoding molecular identification of Chinese medicinal material Radix Paeoniae Rubra (Chishao).

[This corrects the article DOI: 10.3389/fpls.2022.819822.].

Two Multidimensional Chromatography/High-Resolution Mass Spectrometry Approaches Enabling the In-Depth Metabolite Characterization Simultaneously from Three Glycyrrhiza Species: Method Development, Comparison, and Integration.

Two offline multidimensional chromatography/high-resolution mass spectrometry systems (method 1: fractionation and online two-dimensional liquid chromatography, 2D-LC; method 2: fractionation and offline 2D-LC) were established to characterize the metabolites simultaneously from three Glycyrrhiza species. Ion exchange chromatography in the first-dimensional (1D) separation was well fractionated between the acidic (mainly triterpenoids) and weakly acidic components (flavonoids). These obtained subsamples got sophisticated separation by the second (2D) and third dimension (3D) of chromatography either by online reversed-phase chromatography × reversed-phase chromatography (RPC × RPC) or offline hydrophilic interaction chromatography × RPC (HILIC × RPC). Orthogonality for the 2D/3D separations reached 0.73 for method 1 and 0.81 for method 2, respectively. We could characterize 1097 compounds from three Glycyrrhiza species based on an in-house library and 33 reference standards, involving 618 by method 1 and 668 by method 2, respectively. They exhibited a differentiated performance and complementarity in identifying the multiple subclasses of Glycyrrhiza components.

Spatial Distribution and Characterization of the Small-Molecule Metabolites and In Situ Hydrolyzed Oligosaccharides in the Rhizome of Glycyrrhiza uralensis by Desorption Electrospray Ionization-Mass Spectrometry Imaging and High-Resolution Liquid Chromatography-Mass Spectrometry.

Characterization and spatial distribution studies of the metabolome in plants are crucial for revealing the physiology of plants and developing functional foods. Using the rhizome of Glycyrrhiza uralensis as a case, we integrated desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) and high-resolution liquid chromatography/mass spectrometry approaches aimed at characterizing and locating both the small molecules and the macromolecular polysaccharides. Under the optimal conditions, 21 flavonoids and 12 triterpenoids were detected and characterized in different tissues of the rhizome and another 19 components were characterized exclusively by DESI-MSI. Combined with hydrophilic interaction chromatography/ion mobility-quadrupole time-of-flight mass spectrometry, eight different degrees of polymerization of oligosaccharides (after in situ acid hydrolysis) were characterized from the rhizome of G. uralensis. Majority of these metabolites are located in the cortex, phloem, and medulla, which lays the foundation for understanding the physiology of G. uralensis. The useful information can benefit the sustainable utilization and further development of Glycyrrhiza resource.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine: A case study of Amynthas earthworms as "Guang Dilong".

For wild natural medicine, unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials, which affects the efficacy and safety of clinical medication. DNA barcoding as an effective species identification tool is limited by its low sample throughput nature. In this study, combining DNA mini-barcode, DNA metabarcoding and species delimitation method, a novel biological sources consistency evaluation strategy was proposed, and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as "Guang Dilong" and 25 batches of proprietary Chinese medicines. Besides Amynthas aspergillum as the authentic source, 8 other Molecular Operational Taxonomic Units (MOTUs) were elucidated. Significantly, even the subgroups within A. aspergillum revealed here differ significantly on chemical compositions and biological activity. Fortunately, this biodiversity could be controlled when the collection was limited to designated areas, as proved by 2796 "decoction pieces" samples. This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control, and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

Insights into gold-catalyzed formation of aza-heterocycles using benzofuroxans as nitrene transfer reagents: mechanism and origins of chemoselectivity.

Density functional theory (DFT) calculations have been performed to reveal the mechanism of gold(i)-catalyzed annulation of N-allylynamides and benzofuroxans as nitrene transfer reagents to construct azaheterocyclic compounds. The calculated results revealed that the reaction mechanism mainly undergoes eight processes. Among the reaction steps, intramolecular nucleophilic attack of the imino N atom on the α-position of activated gold keteniminium is a rate-determining process, which is different from that proposed previously by experiment. The chemoselectivity of the products is controlled by competition between the cyclopropanation of α-imino gold carbenes and intramolecular nucleophilic attack of the phenyl ring on α-imino gold carbenes, and could be explained by NPA charge. The different yields of cyclopropanated product in different solvents are dictated by the relative polarity leading to the different energy barriers of the rate-determining steps. The present work expounds the experimental observation at the molecular level and is informative for exploring efficient syntheses of 3-azabicyclo[3.1.0]hexanes.

A strategy for a high enrichment of insect mitochondrial DNA for mitogenomic analysis.

Next-generation sequencing has dramatically fostered insect mitogenomic research in recent years. However, studies on the insect mitochondrial genome (mitogenome) assembly mainly rely on the sequencing data from total DNA, which is not cost-effective as a huge data from nuclear DNA are wasted. Besides, many mitogenomic studies require genomic information from individual organisms, whereas the DNA yield from small individual insects is too low to meet the sequencing requirements. Here, we describe a strategy for a high enrichment of insect mitochondrial DNA (mtDNA) using rolling circle amplification (RCA) technique. This strategy consists of standard DNA extraction, RCA enrichment, next-generation sequencing and mitogenome assembly. We have evaluated the performance of this strategy on nine insect species representing eight families of insecta, three other invertebrates, and even two vertebrate specimens. Results show that our strategy is especially suitable for insects, which allows almost all tested insect mtDNA contents to reach 80% and above. A further examination of enrichment efficiency of our strategy among different taxa shows that it is also applicable to other invertebrates and even some vertebrates such as Rhacophorus and ptyas species, although its enrichment efficiency in these groups is lower than that of insects. After treatment with our strategy, small flux sequencing data can realize the assembly of mitogenome with deep coverage, providing a solid base for subsequent mitogenome-based studies.

Biological composition analysis of a natural medicine, Faeces Vespertilionis, with complex sources using DNA metabarcoding.

Faeces Vespertilionis is a commonly used fecal traditional Chinese medicine. Traditionally, it is identified relying only on morphological characters. This poses a serious challenge to the composition analysis accuracy of this complex biological mixture. Thus, for quality control purposes, an accurate and effective method should be provided for taxonomic identification of Faeces Vespertilionis. In this study, 26 samples of Faeces Vespertilionis from ten provinces in China were tested using DNA metabarcoding. Seven operational taxonomic units (OTUs) were detected as belonging to bats. Among them, Hipposideros armiger (Hodgson, 1835) and Rhinolophus ferrumequinum (Schober and Grimmberger, 1997) were the main host sources of Faeces Vespertilionis samples, with average relative abundances of 59.3% and 24.1%, respectively. Biodiversity analysis showed that Diptera and Lepidoptera were the most frequently consumed insects. At the species level, 19 taxa were clearly identified. Overall, our study used DNA metabarcoding to analyze the biological composition of Faeces Vespertilionis, which provides a new idea for the quality control of this special traditional Chinese medicine.

Cinobufacini Injection Inhibits the Proliferation of Triple-Negative Breast Cancer Through the Pin1-TAZ Signaling Pathway.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC), which is characterized by the total absence of human epidermal growth factor receptor 2 (HER2), progesterone receptor (PR), and estrogen receptor (ER) expression. Cinobufacini injection (CI) is the aqueous extract from the dry skin of Bufo gargarizans, which is broadly used for the treatment of malignant tumors. However, the potential mechanism of CI against TNBC has not been fully revealed. In this study, we found that CI inhibited the proliferation of MDA-MB-231 and 4T1 cells in a time- and dose-dependent manner. RNA-seq data showed that downregulated and upregulated genes were mainly enriched in biological processes related to tumor cell proliferation, including cell cycle arrest and regulation of apoptosis signaling pathways. Indeed, after CI treatment, the protein level of CDK1 and Bcl-2/Bax decreased, indicating that CI induced the cell cycle of MDA-MB-231 arrest in the G2/M phase and increased the rate of apoptosis. Meanwhile, CI significantly inhibited the growth of tumor in vivo, and RNA-seq data showed that the TAZ signaling pathway played a vital role after CI treatment. Both immunohistochemistry and Western blot analysis confirmed the downregulation of Pin1 and TAZ, caused by CI treatment. Furthermore, the bioinformatics analysis indicated that Pin1 and TAZ were indeed elevated in TNBC patients, with poor staging, classification, and patient survival rate. In conclusion, CI effectively inhibited the proliferation of TNBC in vitro and in vivo and induced their apoptosis and cycle arrest through the Pin1-TAZ pathway.