Chronic nicotine exposure elicits pain hypersensitivity through activation of dopaminergic projections to anterior cingulate cortex.

BACKGROUND: Cigarette smoking is commonly reported among chronic pain patients in the clinic. Although chronic nicotine exposure is directly linked to nociceptive hypersensitivity in rodents, underlying neurobiological mechanisms remain unknown. METHODS: Multi-tetrode recordings in freely moving mice were used to test the activity of dopaminergic projections from the ventral tegmental area (VTA) to pyramidal neurones in the anterior cingulate cortex (ACC) in chronic nicotine-treated mice. The VTA→ACC dopaminergic pathway was inhibited by optogenetic manipulation to detect chronic nicotine-induced allodynia (pain attributable to a stimulus that does not normally provoke pain) assessed by von Frey monofilaments (force units in g). RESULTS: Allodynia developed concurrently with chronic (28-day) nicotine exposure in mice (0.36 g [0.0141] vs 0.05 g [0.0018], P<0.0001). Chronic nicotine activated dopaminergic projections from the VTA to pyramidal neurones in the ACC, and optogenetic inhibition of VTA dopaminergic terminals in the ACC alleviated chronic nicotine-induced allodynia in mice (0.06 g [0.0064] vs 0.28 g [0.0428], P<0.0001). Moreover, optogenetic inhibition of Drd2 dopamine receptor signalling in the ACC attenuated nicotine-induced allodynia (0.07 g [0.0082] vs 0.27 g [0.0211], P<0.0001). CONCLUSIONS: These findings implicate a role of Drd2-mediated dopaminergic VTA→ACC pathway signalling in chronic nicotine-elicited allodynia.

Analysis of Whole-Genome as a Novel Strategy for Animal Species Identification.

Survival crises stalk many animals, especially endangered and rare animals. Accurate species identification plays a pivotal role in animal resource conservation. In this study, we developed an animal species identification method called Analysis of whole-GEnome (AGE), which identifies species by finding species-specific sequences through bioinformatics analysis of the whole genome and subsequently recognizing these sequences using experimental technologies. To clearly demonstrate the AGE method, Cervus nippon, a well-known endangered species, and a closely related species, Cervus elaphus, were set as model species, without and with published genomes, respectively. By analyzing the whole genomes of C. nippon and C. elaphus, which were obtained through next-generation sequencing and online databases, we built specific sequence databases containing 7,670,140 and 570,981 sequences, respectively. Then, the species specificities of the sequences were confirmed experimentally using Sanger sequencing and the CRISPR-Cas12a system. Moreover, for 11 fresh animal samples and 35 commercially available products, our results were in complete agreement with those of other authoritative identification methods, demonstrating AGE's precision and potential application. Notably, AGE found a mixture in the 35 commercially available products and successfully identified it. This study broadens the horizons of species identification using the whole genome and sheds light on the potential of AGE for conserving animal resources.

Comparative genomics reveal the convergent evolution of CYP82D and CYP706X members related to flavone biosynthesis in Lamiaceae and Asteraceae.

Distant species producing the same secondary metabolites is an interesting and common phenomenon in nature. A classic example of this is scutellarein whose derivatives have been used clinically for more than 30 years. Scutellarein occurs in significant amounts in species of two different orders, Scutellaria baicalensis and Erigeron breviscapus, which diverged more than 100 million years ago. Here, according to the genome-wide selection and functional identification of 39 CYP450 genes from various angiosperms, we confirmed that only seven Scutellaria-specific CYP82D genes and one Erigeron CYP706X gene could perform the catalytic activity of flavone 6-hydroxylase (F6H), suggesting that the convergent evolution of scutellarein production in these two distant species was caused by two independently evolved CYP450 families. We also identified seven Scutellaria-specific CYP82D genes encoding flavone 8-hydroxylase (F8H). The evolutionary patterns of CYP82 and CYP706 families via kingdom-wide comparative genomics highlighted the evolutionary diversity of CYP82D and the specificity of CYP706X in angiosperms. Multi-collinearity and phylogenetic analysis of CYP82D in Scutellaria confirmed that the function of F6H evolved from F8H. Furthermore, the SbaiCYP82D1A319D , EbreCYP706XR130A , EbreCYP706XF312D and EbreCYP706XA318D mutants can significantly decrease the catalytic activity of F6H, revealing the contribution of crucial F6H amino acids to the scutellarein biosynthesis of distant species. This study provides important insights into the multi-origin evolution of the same secondary metabolite biosynthesis in the plant kingdom.

Microglia sustain anterior cingulate cortex neuronal hyperactivity in nicotine-induced pain.

BACKGROUND: Long-term smoking is a risk factor for chronic pain, and chronic nicotine exposure induces pain-like effects in rodents. The anterior cingulate cortex (ACC) has been demonstrated to be associated with pain and substance abuse. This study aims to investigate whether ACC microglia are altered in response to chronic nicotine exposure and their interaction with ACC neurons and subsequent nicotine-induced allodynia in mice. METHODS: We utilized a mouse model that was fed nicotine water for 28 days. Brain slices of the ACC were collected for morphological analysis to evaluate the impacts of chronic nicotine on microglia. In vivo calcium imaging and whole-cell patch clamp were used to record the excitability of ACC glutamatergic neurons. RESULTS: Compared to the vehicle control, the branch endpoints and the length of ACC microglial processes decreased in nicotine-treated mice, coinciding with the hyperactivity of glutamatergic neurons in the ACC. Inhibition of ACC glutamatergic neurons alleviated nicotine-induced allodynia and reduced microglial activation. On the other hand, reactive microglia sustain ACC neuronal excitability in response to chronic nicotine, and pharmacological inhibition of microglia by minocycline or liposome-clodronate reduces nicotine-induced allodynia. The neuron-microglia interaction in chronic nicotine-induced allodynia is mediated by increased expression of neuronal CX3CL1, which activates microglia by acting on CX3CR1 receptors on microglial cells. CONCLUSION: Together, these findings underlie a critical role of ACC microglia in the maintenance of ACC neuronal hyperactivity and resulting nociceptive hypersensitivity in chronic nicotine-treated mice.

Identification of characteristic aroma compounds for spicy in Iris lactea var. chinensis.

Iris lactea var. chinensis (Fisch.) Koidz has a unique floral fragrance that differs from that of other Iris spp.; however, its characteristic aroma composition remains unknown. This study aimed to identify the floral fragrance components of I. lactea var. chinensis during different flowering stages using headspace solid-phase microextraction in conjunction with gas chromatography mass spectrometry, electronic nose, and sensory evaluation. During the three flowering phases (bud stage, bloom stage, and decay stage), 70 volatile organic compounds (VOCs), including 13 aldehydes, 13 esters, 11 alcohols, 10 alkanes, 8 ketones, 7 terpenes, 7 benzenoids, and 1 nitrogenous compound, were identified. According to principal component analysis, the primary VOCs were (-)-pinene, ß-irone, methyl heptenone, phenylethanol, hexanol, and 2-pinene. A comparison of the differential VOCs across the different flowering stages using orthogonal partial least squares discriminant analysis and hierarchical clustering analysis revealed that 3-carene appeared only in the bud stage, whereas hexanol, ethyl caprate, ethyl caproate, linalool, (-)-pinene, and 2-pinene appeared or were present at significantly increased levels during the bloom stage. The phenylethanol, methyl heptenone, 3-methylheptane, and ß-irone reached a peak in the decay stage. The odor activity value and sensory evaluation suggested that "spicy" is the most typical odor of I. lactea var. chinensis, mainly due to 2-methoxy-3-sec-butylpyrazine, which is rare in floral fragrances.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanidin Biosynthesis of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental plant with bulbous flowers noted for their rich blue color. Muscari species have been thought to accumulate delphinidin and cyanidin rather than pelargonidin-type anthocyanins because their dihydroflavonol 4-reductase (DFR) does not efficiently reduce dihydrokaempferol. In our study, we clone a novel DFR gene from blue flowers of Muscari. aucheri. Quantitative real-time PCR (qRT-PCR) and anthocyanin analysis showed that the expression pattern of MaDFR had strong correlations with the accumulation of delphinidin, relatively weak correlations with cyanidin, and no correations with pelargonidin. However, in vitro enzymatic analysis revealed that the MaDFR enzyme can reduce all the three types of dihydroflavonols (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), although it most preferred dihydromyricetin as a substrate to produce leucodelphinidin, the precursor of blue-hued delphinidin. This indicated that there may be other functional genes responsible for the loss of red pelargonidin-based pigments in Muscari. To further verify the substrate-specific selection domains of MaDFR, an assay of amino acid substitutions was conducted. The activity of MaDFR was not affected whenever the N135 or E146 site was mutated. However, when both of them were mutated, the catalytic activity of MaDFR was lost completely. The results suggest that both the N135 and E146 sites are essential for the activity of MaDFR. Additionally, the heterologous expression of MaDFR in tobacco (Nicotiana tabacum) resulted in increasing anthocyanin accumulation, leading to a darker flower color, which suggested that MaDFR was involved in color development in flowers. In summary, MaDFR has a high preference for dihydromyricetin, and it could be a powerful candidate gene for genetic engineering for blue flower colour modification. Our results also make a valuable contribution to understanding the basis of color variation in the genus Muscari.

Differential Regulation of Anthocyanins in Green and Purple Turnips Revealed by Combined De Novo Transcriptome and Metabolome Analysis.

Purple turnip Brassica rapa ssp. rapa is highly appreciated by consumers but the metabolites and molecular mechanisms underlying the root skin pigmentation remain open to study. Herein, we analyzed the anthocyanin composition in purple turnip (PT) and green turnip (GT) at five developmental stages. A total of 21 anthocyanins were detected and classified into the six major anthocynanin aglycones. Distinctly, PT contains 20 times higher levels of anthocyanins than GT, which explain the difference in the root skin pigmentation. We further sequenced the transcriptomes and analyzed the differentially expressed genes between the two turnips. We found that PT essentially diverts dihydroflavonols to the biosynthesis of anthocyanins over flavonols biosynthesis by strongly down-regulating one flavonol synthase gene, while strikingly up-regulating dihydroflavonol 4-reductase (DFR), anthocyanidin synthase and UDP-glucose: flavonoid-3-O-glucosyltransferase genes as compared to GT. Moreover, a nonsense mutation identified in the coding sequence of the DFR gene may lead to a nonfunctional protein, adding another hurdle to the accumulation of anthocyanin in GT. We also uncovered several key members of MYB, bHLH and WRKY families as the putative main drivers of transcriptional changes between the two turnips. Overall, this study provides new tools for modifying anthocyanin content and improving turnip nutritional quality.

Anthocyanin Profiles in Flowers of Grape Hyacinth.

Grape hyacinth (Muscari spp.) is a popular ornamental bulbous perennial famous for its blue flowers. To understand the chemical basis of the rich blue colors in this plant, anthocyanin profiles of six blue flowering grape hyacinths as well as one pink and one white cultivar were determined using high-performance liquid chromatography and mass spectrometry. Along with two known compounds, eight putative anthocyanins were identified in the tepals of grape hyacinth for the first time. The accumulation and distribution of anthocyanins in the plant showed significant cultivar and flower development specificity. Violet-blue flowers mainly contained simple delphinidin-type anthocyanins bearing one or two methyl-groups but no acyl groups, whereas white and pink flowers synthesised more complex pelargonidin/cyanidin-derivatives with acyl-moieties but no methyl-groups. The results partially reveal why solid blue, orange or red flowers are rare in this plant in nature. In addition, pelargonidin-type anthocyanins were found for the first time in the genus, bringing more opportunities in terms of breeding of flower color in grape hyacinth.

Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth.

Grape hyacinth (Muscari) is an important ornamental bulbous plant with an extraordinary blue colour. Muscari armeniacum, whose flowers can be naturally white, provides an opportunity to unravel the complex metabolic networks underlying certain biochemical traits, especially colour. A blue flower cDNA library of M. armeniacum and a white flower library of M. armeniacum f. album were used for transcriptome sequencing. A total of 89 926 uni-transcripts were isolated, 143 of which could be identified as putative homologues of colour-related genes in other species. Based on a comprehensive analysis relating colour compounds to gene expression profiles, the mechanism of colour biosynthesis was studied in M. armeniacum. Furthermore, a new hypothesis explaining the lack of colour phenotype of the grape hyacinth flower is proposed. Alteration of the substrate competition between flavonol synthase (FLS) and dihydroflavonol 4-reductase (DFR) may lead to elimination of blue pigmentation while the multishunt from the limited flux in the cyanidin (Cy) synthesis pathway seems to be the most likely reason for the colour change in the white flowers of M. armeniacum. Moreover, mass sequence data obtained by the deep sequencing of M. armeniacum and its white variant provided a platform for future function and molecular biological research on M. armeniacum.

Unveiling Fall Triggers in Older Adults: A Machine Learning Graphical Model Analysis.

While existing research has identified diverse fall risk factors in adults aged 60 and older across various areas, comprehensively examining the interrelationships between all factors can enhance our knowledge of complex mechanisms and ultimately prevent falls. This study employs a novel approach-a mixed undirected graphical model (MUGM)-to unravel the interplay between sociodemographics, mental well-being, body composition, self-assessed and performance-based fall risk assessments, and physical activity patterns. Using a parameterized joint probability density, MUGMs specify the higher-order dependence structure and reveals the underlying graphical structure of heterogeneous variables. The MUGM consisting of mixed types of variables (continuous and categorical) has versatile applications that provide innovative and practical insights, as it is equipped to transcend the limitations of traditional correlation analysis and uncover sophisticated interactions within a high-dimensional data set. Our study included 120 elders from central Florida whose 37 fall risk factors were analyzed using an MUGM. Among the identified features, 34 exhibited pairwise relationships, while COVID-19-related factors and housing composition remained conditionally independent from all others. The results from our study serve as a foundational exploration, and future research investigating the longitudinal aspects of these features plays a pivotal role in enhancing our knowledge of the dynamics contributing to fall prevention in this population.

Identification of closely related species in Aspergillus through Analysis of Whole-Genome.

The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/µl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.

Analysis of Whole-Genome facilitates rapid and precise identification of fungal species.

Fungal identification is a cornerstone of fungal research, yet traditional molecular methods struggle with rapid and accurate onsite identification, especially for closely related species. To tackle this challenge, we introduce a universal identification method called Analysis of whole GEnome (AGE). AGE includes two key steps: bioinformatics analysis and experimental practice. Bioinformatics analysis screens candidate target sequences named Targets within the genome of the fungal species and determines specific Targets by comparing them with the genomes of other species. Then, experimental practice using sequencing or non-sequencing technologies would confirm the results of bioinformatics analysis. Accordingly, AGE obtained more than 1,000,000 qualified Targets for each of the 13 fungal species within the phyla Ascomycota and Basidiomycota. Next, the sequencing and genome editing system validated the ultra-specific performance of the specific Targets; especially noteworthy is the first-time demonstration of the identification potential of sequences from unannotated genomic regions. Furthermore, by combining rapid isothermal amplification and phosphorothioate-modified primers with the option of an instrument-free visual fluorescence method, AGE can achieve qualitative species identification within 30 min using a single-tube test. More importantly, AGE holds significant potential for identifying closely related species and differentiating traditional Chinese medicines from their adulterants, especially in the precise detection of contaminants. In summary, AGE opens the door for the development of whole-genome-based fungal species identification while also providing guidance for its application in plant and animal kingdoms.

Protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step immunoprecipitation approach.

Co-immunoprecipitation (coIP) is an experimental technique to study protein-protein interactions (PPIs). However, single-step coIP can only be used to identify the interaction between two proteins and does not solve the interaction testing of ternary complexes. Here, we present a protocol to test for the formation of ternary protein complexes in vivo or in vitro using a two-step coIP approach. We describe steps for cell culture and transfection, elution of target proteins, and two-step coIP including western blot analyses. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Construction of a high-efficiency GjCCD4a mutant and its application for de novo biosynthesis of five crocins in Escherichia coli.

Crocins are bioactive natural products that rarely exist in plants. High costs and resource shortage severely limit its development and application. Synthetic biology studies on crocins are of considerable global interest. However, the lack of high-efficiency genetic tools and complex cascade biocatalytic systems have substantially hindered progress in crocin biosynthesis-related research. Based on mutagenesis, a high-efficiency GjCCD4a mutant (N212m) was constructed with a catalytic efficiency that was 25.08-fold higher than that of the wild-type. Solubilized GjCCD4a was expressed via fusion with an MBP tag. Moreover, N212m and ten other genes were introduced into Escherichia coli for the de novo biosynthesis of five crocins. The engineered E57 strain produced crocins III and V with a total yield of 11.50 mg/L, and the E579 strain produced crocins I-V with a total output of 8.43 mg/L at shake-flask level. This study identified a marvelous genetic element (N212m) for crocin biosynthesis and achieved its de novo biosynthesis in E. coli using glucose. This study provides a reference for the large-scale production of five crocins using E. coli cell factories.

ATG5 nonautophagically regulates inflammation and differentiation in mouse embryonic stem cells.

Embryonic stem cells (ESCs), with abilities of infinite proliferation (self-renewal) and to differentiate into distinct cell types (pluripotency), show attenuated inflammatory response against cytokines or pathogens, which is recognized as a unique characteristic of ESCs compared with somatic cells. However, the underlying molecular mechanisms remain unclear, and whether the attenuated inflammatory state is involved in ESC differentiation is completely unknown. Our recent study demonstrated that macroautophagy/autophagy-related protein ATG5 inhibits the inflammatory response of mouse ESCs (MmESCs) by promoting the degradation of BTRC/ß-TrCP1 and further the downregulation of NFKB/NF-κB signaling. In addition, maintenance of an attenuated inflammation status in MmESCs is required for their differentiation. In conclusion, ATG5 is a key regulator for the regulation of inflammatory response and differentiation of MmESCs.

Angiotensin Type 2 Receptor Pharmacological Agonist Relieves Neurocognitive Deficits via Reducing Neuroinflammation and Microglial Engulfment of Dendritic Spines.

Mechanically ventilated patients suffering critical illness are at high risk of developing neurocognitive impairments. Angiotensin type 2 receptor (AGTR2) has been demonstrated to be anti-inflammatory and neuroprotective. The present study thus aimed to investigate whether AGTR2 can alleviate cerebral dysfunction in mice subjected to cochallenge with lipopolysaccharide (LPS) and mechanical ventilation (MV), and to reveal the underlying mechanism. We utilized a mice model that received a single injection of LPS (1 mg/kg, intraperitoneally) followed 2 h later by MV (10 ml/kg, lasting for 2 h). Pretreatment with the AGTR2 pharmacological agonist C21 (0.03, 0.3, and 3 mg/kg, intraperitoneally, once daily, lasting for 10 days). Locomotor activity and behavioral deficits were evaluated 24 h post-MV by open-field and fear-condition tests. Brain hippocampus and prefrontal cortex tissues were collected for immunofluorescence staining and western blotting to evaluate the resulting impacts on microglia, including morphological traits, functional markers, synaptic engulfment, superoxide production, and signaling molecules. Compared with vehicle-control, pre-administrated C21 reduced the branch endpoints and length of microglia processes in a dose-dependent manner in mice subjected to LPS/MV. The neuroprotective effect of AGTR2 was behaviorally confirmed by the improvement of memory decline in LPS/MV-treated mice following C21 pretreatment. In addition to morphological alterations, C21 reduced microglial functional markers and reduced microglial-dendrite contact and microglial engulfment of synaptic protein markers. In terms of the underlying molecular mechanism, AGTR2 stimulation by C21 leads to activation of protein phosphatase 2A, which subsequently mitigates microglial PKCδ and NF-κB activation, and inhibites NOX2-derived ROS production. The AGTR2 agonist C21 alleviates behavioral deficits in those mice subjected to LPS/MV, via mechanisms that involve reactive microglia and abnormal synaptic plasticity in NOX2-derived ROS and the PKCδ-NFκB pathway.

TaqMan Probe-Based Quantitative Real-Time PCR to Detect Panax notoginseng in Traditional Chinese Patent Medicines.

Background: There has been global concern about the safety and accuracy of traditional Chinese patent medicines (TCPMs). Panax notoginseng, also known as sanqi, is an important constituent of TCPMs. However, identifying the species contained in TCPMs is challenging due to the presence of multiple ingredients and the use of various preparation processes. Objective: To detect P. notoginseng in TCPMs. Methods: A TaqMan probe-based qPCR assay was constructed and validated with DNA extracted from P. notoginseng and adulterants. In total, 75 samples derived from 25 batches of TCPMs were tested using the constructed qPCR method. Results: A TaqMan probe-based qPCR assay targeting P. notoginseng was established. The constructed qPCR assay could specifically discriminate P. notoginseng from Panax ginseng, Panax quinquefolium and Curcuma aromatica Salisb. cv. Wenyujin. The sensitivity study showed that the detectable DNA template concentration of P. notoginseng for this qPCR assay was 0.001 ng/µl. All 75 samples from TCPMs were confirmed to contain P. notoginseng by the qPCR assay. Conclusions: The qPCR method can accurately identify P. notoginseng in TCPMs and is promising as a powerful tool for quality control and market regulation.

[Pollution Characteristics and Driving Factors of Antibiotic Resistance Genes in Dexing Copper Mine].

Environmental antibiotic resistance genes (ARGs) are a type of emerging pollutant that has been widely concerning. However, investigations into the contamination of ARGs in mining areas have been scarce. Here, the types, abundances, and influencing factors of ARGs and mobile genetic elements (MGEs) were investigated in soil/sediment of the Dexing copper mine area in June 2019 by using high-throughput quantitative polymerase chain reaction (HT-qPCR). Furthermore, the influence of heavy metals and MGEs factors on ARGs was studied using the multivariate statistical analysis method. The results showed that there were a variety of ARGs in the Dexing copper mining area, and the maximum detected number of ARGs was 70. At the relative abundance level, the relative abundance of individual sites reached 0.085. In the Dexing copper mine, multidrug, MLSB, ß-lactamases, tetracycline, and aminoglycoside resistance genes were the dominant ARG classes based on their numbers. The efflux pump was the most dominant resistance mechanism, followed by antibiotic deactivation and cellular protection. There was a significant positive correlation between the abundance of ARGs and MGEs (P<0.05), and TnpA04 and Inti1 were the most important MEGs in Dexing copper mine samples, indicating that horizontal gene transfer might be an important mechanism for the spread of environmental ARGs. The results of Pearson correlation analysis and RDA analysis showed that the content of Cu was significantly positively correlated with the detected numbers and abundance of ARGs (P<0.05), suggesting that the high content of Cu in the Dexing copper mining area might be an important driving factor for the formation of ARGs.

Droplet digital PCR for the identification of plant-derived adulterants in highly processed products.

BACKGROUND: The high sensitivity of droplet digital PCR (ddPCR) contributes to its excellent performance in animal and microorganism identification, but the utilization of ddPCR is limited in plant adulterant identification of highly processed products for which effective methods are lacking. PURPOSE: This study investigated the feasibility of ddPCR in the identification of plant adulterants in Chinese patent medicine (CPM) as groundwork to develop ddPCR assays for other highly processed goods. METHODS: The original plant, processed and highly processed products of Mutong (Akebiae Caulis) and its two adulterants were used to analyze the specificity, sensitivity, and practical performance of the developed singleplex and triplex ddPCR assays. RESULTS: The results revealed that the limit of detection (LOD) and limit of quantification (LOQ) for the selective ddPCR assays developed to identify Mutong and its adulterants were 0.00002 ng/µl and 0.00016 ng/µl, respectively, and that the regression equations representing the relationships between DNA concentration and target copy number all exhibited good linearity. Furthermore, the common adulterant of Mutong in three samples of Longdan Xiegan pills was successfully identified through ddPCR assays and confirmed by Sanger sequencing. CONCLUSION: This work comprehensively revealed the great ability of ddPCR technology in detecting plant adulterants in traditional Chinese medicine (TCM), providing a method for the quality control of highly processed plant products with complex components for commonly used goods.

Application of DNA barcoding to the entire traditional Chinese medicine industrial chain: A case study of Rhei Radix et Rhizoma.

BACKGROUND: Safety concerns, caused by complex and unpredictable adulterants, run through the entire industrial chain of traditional Chinese medicines (TCMs). However, the conventional circulation traceability system only focuses on a certain end or link at the back end of the TCM industrial chain, ignoring the integrity of the links cross the entire industrial chain and lacking traceability. In consequence, a strict and rational supervision system is urgently required for the entire industrial chain. HYPOTHESIS/PURPOSE: We hypothesize that DNA barcoding would be a suitable measure for the traceability of adulterants in the entire TCM industrial chain. METHODS: In this study, Rhei Radix et Rhizoma was selected as a model to establish a traceability system for the entire TCM industrial chain. A total of 110 samples, including leaves, seeds, roots, decoction pieces, and traditional Chinese patent medicines (TCPMs), were collected upstream, midstream, and downstream of the entire industrial chain of Rhei Radix et Rhizoma. The ndhF-rpl32 fragment rather than the universal DNA barcodes, which could not distinguish the three original species of Rhei Radix et Rhizoma, was selected as a specific DNA barcode to evaluate the practical application of DNA barcoding in the chain. RESULTS: The results showed that the ndhF-rpl32 fragment in all samples could be amplified and bi-directionally sequenced. Based on the standard operating procedures of DNA barcoding, the ndhF-rpl32 fragment clearly distinguished the seven Rheum species collected upstream of the entire industrial chain. For the samples collected midstream and downstream of the entire industrial chain, 25% of the 36 commercial decoction pieces samples were identified as adulterants, whereas the eight TCPM samples were all derived from genuine Rhei Radix et Rhizoma. CONCLUSIONS: This study shows that DNA barcoding is a powerful and suitable technology that can be applied to trace TCMs in the entire industrial chain, thereby assuring clinical medication safety.