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.

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.

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.

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.

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.

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.

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.

Application of Large-Scale Molecular Prediction for Creating the Preferred Precursor Ions List to Enhance the Identification of Ginsenosides from the Flower Buds of Panax ginseng.

This work was designed to evaluate the coverage of data-dependent acquisition (DDA) extensively utilized in the untargeted metabolite/component identification in the food sciences and pharmaceutical analysis. Using saponins from the flower buds of Panax ginseng (PGF) as an example, precursor ions list (PIL)-including DDA on a Q-Orbitrap mass spectrometer could enable higher coverage than the other four MS2 acquisition approaches in characterizing PGF ginsenosides. A "Virtual Library of Ginsenoside" containing 13,536 ginsenoside molecules was established by C-language-programmed large-scale molecular prediction, which in combination with mass defect filtering could create a new PIL involving 1859 PGF saponin precursors. We could newly obtain the MS2 spectra of at least 17 components and characterize 36 ginsenosides with unknown masses, among the 164 compounds identified from PGF. Conclusively, a molecular-prediction-oriented PIL in DDA can assist to discover more potentially novel molecules benefiting to the development of functional foods and new drugs.

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.

Development of Mini-Barcode Based on Chloroplast Genome and Its Application in Metabarcoding Molecular Identification of Chinese Medicinal Material Radix Paeoniae Rubra (Chishao).

Radix Paeoniae Rubra (Chishao), a typical multi-origin Chinese medicinal material, originates from the dried roots of Paeonia lactiflora or P. veitchii. The previous study suggested that these two commonly used Chishao showed variation in their chemical compositions and clinical efficacies. Therefore, accurate identification of different Chishao species was of great significance for the guide of clinical medication, and timely treatment of patients. In this study, the chloroplast genome sequences of P. lactiflora and P. veitchii were obtained by next-generation sequencing (NGS) technology, and then the hypervariable regions were selected to design two mini-barcode candidates for species identification. Combined with DNA metabarcoding technology, we performed qualitative and quantitative analysis on the artificially mixed samples of P. lactiflora and P. veitchii and evaluated the identification ability of these mini-barcode candidates. Furtherly, the mini-barcode with good performance was applied to distinguish the Chinese patent medicine "cerebral thrombosis tablets" containing Chishao. The results indicated that the chloroplast genomes of P. lactiflora and P. veitchii were 152,750 and 152,527 bp, respectively. As published previously, they exhibited a typical quadripartite structure including a large single-copy region (LSC), a small single-copy region (SSC) and a pair of inverted repeat regions (IRs). The nucleotide polymorphism analysis revealed seven variable protein-coding regions as petL, psaI, psbJ, rpl16, ycf1b, psaC, and ndhF, and two mini-barcodes were developed from ycf1b and ndhF respectively. The result suggested that both two mini-barcodes performed well distinguishing P. lactiflora from P. veitchii. Besides, P. lactiflora was the only raw material of Chishao in all collected "cerebral thrombosis tablets" samples. In general, this study has established a method to realize the qualitative and quantitative identification of Chishao as multi-origin Chinese medicinal materials, which can be applied to Chinese patent medicines containing Chishao.

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.

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.

Tetrandrine promotes angiogenesis via transcriptional regulation of VEGF-A.

Angiogenesis is crucial for tissue damage repair in ischemic cardiovascular diseases. Vascular endothelial growth factor A (VEGF-A) acts as a vital mediator in angiogenesis. In this study, tetrandrine (Tet) was found from 23 herbal chemicals to increase VEGF-A mRNA expression in H9c2 cells and the effect was confirmed in freshly isolated neonatal rat cardiomyocytes. The effect of Tet on VEGF-A expression and the possible mechanism were investigated. Tet treatment increased de novo VEGF-A mRNA synthesis and did not affect VEGF-A mRNA stability. The circulating chromosome conformation capture (4C) experiments indicated that Tet enhanced VEGF-A transcription by targeting a regulatory element beyond the 2.6 kb region of the translation start site. Tet augmented the angiogenic activities of endothelial cells. It also enhanced blood flow restoration and capillary vessel density following ischemic limb injury associated with an escalation of VEGF-A expression. Moreover, in myocardial infarction (MI) model Tet treatment elevated neovascularization, reduced infarction size, and improved heart function via upregulating VEGF-A levels. Our results suggested that Tet increased VEGF-A transcription through a novel mechanism that likely involves a distant regulatory element and may be useful for therapeutic angiogenesis for ischemic diseases.

Investigation on the stability in plant metabolomics with a special focus on freeze-thaw cycles: LC-MS and NMR analysis to Cassiae Semen (Cassia obtusifolia L.) seeds as a case study.

Metabolomics is a rapid and sensitive tool for the detection of dynamic metabolic compositions in the study of systemic metabolic consequences. However, it is also susceptible to a tiny variation of pre-analytical handling procedures. To provide reproducible results, specific knowledge on metabolites perturbance along with different freeze-thaw cycles (FTCs) is needed for further metabolomics studies. In this paper, five FTCs of germinated Cassiae Semen (CS) were chosen as a case study to investigate the influence of FTC effect based on UHPLC-Q-Orbitrap-MS and NMR technologies. A total of 108 metabolites were relatively quantified by LC-MS and NMR analyses. Principal component analysis (PCA) showed that the first and second FTC samples are welly separated from the other groups; however, the extent of FTC-induced effects are smaller after the third cycle. Upon five consecutive FTCs, alterations which consisted of decreased stachyose, sucrose, norrubrofusarin-6-O-ß-d-glucopyranoside, and quercetin 3-(3″-acetylgalactoside), as well as increased phenylalanine, leucine, isoleucine, methionine, phenylalanine, mannose, gluconic acid, and valine, could be observed. FTC does not exert the same effect on all metabolites. Although a large number of secondary metabolites were stable when subjected to five FTCs, FTC effects may lead to false-positive in the discovery of biomarker. In the case of reusing plant seed samples, no more than three consecutive freeze-thaw cycles were found advisable. This work provides unique perspectives on the FTC effects, which may fill in some existing gaps in the knowledge of the stability of plant metabolites during sample pre-handling.

Insights into molecular structure, genome evolution and phylogenetic implication through mitochondrial genome sequence of Gleditsia sinensis.

Gleditsia sinensis is an endemic species widely distributed in China with high economic and medicinal value. To explore the genomic evolution and phylogenetic relationships of G. sinensis, the complete mitochondrial (mt) genome of G. sinensis was sequenced and assembled, which was firstly reported in Gleditsia. The mt genome was circular and 594,121 bp in length, including 37 protein-coding genes (PCGs), 19 transfer RNA (tRNA) genes and 3 ribosomal RNA (rRNA) genes. The overall base composition of the G. sinensis mt genome was 27.4% for A, 27.4% for T, 22.6% for G, 22.7% for C. The comparative analysis of PCGs in Fabaceae species showed that most of the ribosomal protein genes and succinate dehydrogenase genes were lost. In addition, we found that the rps4 gene was only lost in G. sinensis, whereas it was retained in other Fabaceae species. The phylogenetic analysis based on shared PCGs of 24 species (22 Fabaceae and 2 Solanaceae) showed that G. sinensis is evolutionarily closer to Senna species. In general, this research will provide valuable information for the evolution of G. sinensis and provide insight into the phylogenetic relationships within the family Fabaceae.

Leveraging 16S rRNA Microbiome Sequencing Data to Identify Bacterial Signatures for Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a chronic gastrointestinal disorder characterized by abdominal pain or discomfort. Previous studies have illustrated that the gut microbiota might play a critical role in IBS, but the conclusions of these studies, based on various methods, were almost impossible to compare, and reproducible microorganism signatures were still in question. To cope with this problem, previously published 16S rRNA gene sequencing data from 439 fecal samples, including 253 IBS samples and 186 control samples, were collected and processed with a uniform bioinformatic pipeline. Although we found no significant differences in community structures between IBS and healthy controls at the amplicon sequence variants (ASV) level, machine learning (ML) approaches enabled us to discriminate IBS from healthy controls at genus level. Linear discriminant analysis effect size (LEfSe) analysis was subsequently used to seek out 97 biomarkers across all studies. Then, we quantified the standardized mean difference (SMDs) for all significant genera identified by LEfSe and ML approaches. Pooled results showed that the SMDs of nine genera had statistical significance, in which the abundance of Lachnoclostridium, Dorea, Erysipelatoclostridium, Prevotella 9, and Clostridium sensu stricto 1 in IBS were higher, while the dominant abundance genera of healthy controls were Ruminococcaceae UCG-005, Holdemanella, Coprococcus 2, and Eubacterium coprostanoligenes group. In summary, based on six published studies, this study identified nine new microbiome biomarkers of IBS, which might be a basis for understanding the key gut microbes associated with IBS, and could be used as potential targets for microbiome-based diagnostics and therapeutics.

The complete mitochondrial genome sequence of Asclepios apicalis (Gerromorpha: Gerridae).

In this study, the complete mitochondrial genome of Asclepios apicalis was sequenced and assembled, which was first reported in Asclepios. The mitogenome of Asclepios apicalis was 15,391 bp in length, and it contained 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, 13 protein-coding genes (PCGs), and a control region (D-loop), the overall base nucleotide compositions encoded was 42.9% A, 14.3% C, 10.0% G, and 32.8% T.

The complete mitochondrial genome sequence of Aquarius elongatus (Hemiptera: Gerridae).

In this study, we report the complete mitochondrial genome of Aquarius elongatus. The mitogenome was 15,370 bp in length, comprising 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. Maximum-likelihood (ML) phylogenetic tree indicated that Aquarius elongatus has a close relationship with Aquarius paludum. In general, this study provides meaningful genetic information for A. elongatus.