Functional characterization of four mono-terpene synthases (TPSs) provided insight into the biosynthesis of volatile monoterpenes in the medicinal herb Blumea balsamifera.

Blumea balsamifera, a wooden plant belonging to the family Asteraceae, is a medicinal herb with anticancer, antiviral, and multiple pharmacological effects, which are believed to be caused by its essential oil. The essential oil from B. balsamifera is comprised of mono- and sesqui-terpenes as the majority. Unfortunately, this plant has been facing the challenge of resource shortage, which could be effectively alleviated by biological engineering. Therefore, the identification of key elements involved in the biosynthesis of active ingredients becomes an indispensable prerequisite. In this study, candidate genes encoding monoterpene synthase were screened by transcriptome sequencing combined with metabolomics profiling in the roots, stems, and leaves of B. balsamifera. Then, these candidates were successfully cloned and verified by heterologous expression and in vitro enzyme activity assays. As a result, six candidate BbTPS genes were isolated from B. balsamifera, of which three encoded single-product monoterpene synthases and one encoded a multi-product monoterpene synthase. Among them, BbTPS1, BbTPS3, and BbTPS4 could catalyze the formation of D-limonene, α-phellandrene, and L-borneol, respectively. Meanwhile, BbTPS5 functioned in catalyzing GPP into terpinol, ß-phellandrene, ß-myrcene, D-limonene, and 2-carene in vitro. In general, our results provided important elements for the synthetic biology of volatile terpenes in B. balsamifera, which laid a foundation for subsequent heterologous production of these terpenoids through metabolic engineering and increasing their yield, as well as promoting sustainable development and utilization of B. balsamifera. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01306-8.

Comprehensive analysis of complete chloroplast genome and phylogenetic aspects of ten Ficus species.

BACKGROUND: The large genus Ficus comprises approximately 800 species, most of which possess high ornamental and ecological values. However, its evolutionary history remains largely unknown. Plastome (chloroplast genome) analysis had become an essential tool for species identification and for unveiling evolutionary relationships between species, genus and other rank groups. In this work we present the plastomes of ten Ficus species. RESULTS: The complete chloroplast (CP) genomes of eleven Ficus specimens belonging to ten species were determined and analysed. The full length of the Ficus plastome was nearly 160 kbp with a similar overall GC content, ranging from 35.88 to 36.02%. A total of 114 unique genes, distributed in 80 protein-coding genes, 30 tRNAs, and 4 rRNAs, were annotated in each of the Ficus CP genome. In addition, these CP genomes showed variation in their inverted repeat regions (IR). Tandem repeats and mononucleotide simple sequence repeat (SSR) are widely distributed across the Ficus CP genome. Comparative genome analysis showed low sequence variability. In addition, eight variable regions to be used as potential molecular markers were proposed for future Ficus species identification. According to the phylogenetic analysis, these ten Ficus species were clustered together and further divided into three clades based on different subgenera. Simultaneously, it also showed the relatedness between Ficus and Morus. CONCLUSION: The chloroplast genome structure of 10 Ficus species was similar to that of other angiosperms, with a typical four-part structure. Chloroplast genome sizes vary slightly due to expansion and contraction of the IR region. And the variation of noncoding regions of the chloroplast genome is larger than that of coding regions. Phylogenetic analysis showed that these eleven sampled CP genomes were divided into three clades, clustered with species from subgenus Urostigma, Sycomorus, and Ficus, respectively. These results support the Berg classification system, in which the subgenus Ficus was further decomposed into the subgenus Sycomorus. In general, the sequencing and analysis of Ficus plastomes, especially the ones of species with no or limited sequences available yet, contribute to the study of genetic diversity and species evolution of Ficus, while providing useful information for taxonomic and phylogenetic studies of Ficus.

Metabolic stimulation-elicited transcriptional responses and biosynthesis of acylated triterpenoids precursors in the medicinal plant Helicteres angustifolia.

BACKGROUND: Helicteres angustifolia has long been used in Chinese traditional medicine. It has multiple pharmacological benefits, including anti-inflammatory, anti-viral and anti-tumor effects. Its main active chemicals include betulinic acid, oleanolic acid, helicteric acid, helicterilic acid, and other triterpenoid saponins. It is worth noting that some acylated triterpenoids, such as helicteric acid and helicterilic acid, are characteristic components of Helicteres and are relatively rare among other plants. However, reliance on natural plants as the only sources of these is not enough to meet the market requirement. Therefore, the engineering of its metabolic pathway is of high research value for enhancing the production of secondary metabolites. Unfortunately, there are few studies on the biosynthetic pathways of triterpenoids in H. angustifolia, hindering its further investigation. RESULTS: Here, the RNAs of different groups treated by metabolic stimulation were sequenced with an Illumina high-throughput sequencing platform, resulting in 121 gigabases of data. A total of 424,824 unigenes were obtained after the trimming and assembly of the raw data, and 22,430 unigenes were determined to be differentially expressed. In addition, three oxidosqualene cyclases (OSCs) and four Cytochrome P450 (CYP450s) were screened, of which one OSC (HaOSC1) and one CYP450 (HaCYPi3) achieved functional verification, suggesting that they could catalyze the production of lupeol and oleanolic acid, respectively. CONCLUSION: In general, the transcriptomic data of H. angustifolia was first reported and analyzed to study functional genes. Three OSCs, four CYP450s and three acyltransferases were screened out as candidate genes to perform further functional verification, which demonstrated that HaOSC1 and HaCYPi3 encode for lupeol synthase and ß-amyrin oxidase, which produce corresponding products of lupeol and oleanolic acid, respectively. Their successful identification revealed pivotal steps in the biosynthesis of acylated triterpenoids precursors, which laid a foundation for further study on acylated triterpenoids. Overall, these results shed light on the regulation of acylated triterpenoids biosynthesis.

Characteristics analysis of the complete Wurfbainia villosa chloroplast genome.

Wurfbainia villosa, which belongs to the huge family Zingiberaceae, is used in the clinic for the treatment of spleen and stomach diseases in southern China. The complete chloroplast genome of W. villosa was sequenced and analyzed using next-generation sequencing technology in the present work. The results showed that the W. villosa chloroplast genome is a circular molecule with 163,608 bp in length. It harbors a pair of inverted repeat regions (IRa and IRb) of 29,820 bp in length, which separate the large single copy (LSC, 88,680 bp) region and the small single copy (SSC, 15,288 bp) region. After annotation, 134 genes were identified in this plastome in total, comprising of 87 protein-coding genes, 38 transfer RNA genes, 8 ribosomal RNA genes and one pseudogene (ycf1). Codon usage, RNA editing sites and single/long sequence repeats were investigated to understand the structural characteristics of the W. villosa chloroplast genome. Furthermore, IR contraction and expansion were analyzed by comparison of complete chloroplast genomes of W. villosa and four other Zingiberaceae species. Finally, a phylogeny study based on the chloroplast genome of W. villosa, along with that of 15 different species, was conducted to further investigate the relationship among these lineages. Overally, our results represented the first insight into the chloroplast genome of W. villosa, and could serve as a significant reference for species identification, genetic diversity analysis and phylogenetic research between W. villosa and other species within Zingiberaceae.

[Preliminary study on effect of Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces on intestinal flora of mice].

This study aims to analyze and compare the effect of cell wall-broken decoction pieces, conventional decoction pieces and conventional powder of Rhodiolae Crenulatae Radix et Rhizoma on the intestinal flora of normal mice. The conventional bacterial culture and PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) were adopted for the mice after the oral administration for 14 days. According to the bacterial culture results, the 1/8 dose cell wall-broken decoction pieces group showed fewer Enterococcus and Escherichia coli bacillus but more Lactobacillus and Bifidobacterium than the conventional decoction pieces group and the traditional powder group (P <0.05). Meanwhile, on the basis of the PCR-DGGE results, the 1/8 dose cell wall-broken decoction pieces group revealed the highest Shannon-Wiener index (H) and species richness (S) among the seven groups, with extremely significant differences compared with the normal group (P <0.01), significant differences compared with the conventional decoction pieces group and the conventional powder group (P <0.05) and a high intra-group similarity. In conclusion, the long-term intake of 1/8 dose Rhodiolae Crenulatae Radix et Rhizoma cell wall-broken decoction pieces showed a certain effect in regulating intestinal tract by promoting the growth of Lactobacillus and Bifidobacterium. Furthermore, the intestinal flora community will become more stable.

Exploring the mechanism of 6-Methoxydihydrosanguinarine in the treatment of lung adenocarcinoma based on network pharmacology, molecular docking and experimental investigation.

BACKGROUND: 6-Methoxydihydrosanguinarine (6-MDS) has shown promising potential in fighting against a variety of malignancies. Yet, its anti­lung adenocarcinoma (LUAD) effect and the underlying mechanism remain largely unexplored. This study sought to explore the targets and the probable mechanism of 6-MDS in LUAD through network pharmacology and experimental validation. METHODS: The proliferative activity of human LUAD cell line A549 was evaluated by Cell Counting Kit-8 (CCK8) assay. LUAD related targets, potential targets of 6-MDS were obtained from databases. Venn plot analysis were performed on 6-MDS target genes and LUAD related genes to obtain potential target genes for 6-MDS treatment of LUAD. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was utilized to perform a protein-protein interaction (PPI) analysis, which was then visualized by Cytoscape. The hub genes in the network were singled out by CytoHubba. Metascape was employed for GO and KEGG enrichment analyses. molecular docking was carried out using AutoDock Vina 4.2 software. Gene expression levels, overall survival of hub genes were validated by the GEPIA database. Protein expression levels, promotor methylation levels of hub genes were confirmed by the UALCAN database. Timer database was used for evaluating the association between the expression of hub genes and the abundance of infiltrating immune cells. Furthermore, correlation analysis of hub genes expression with immune subtypes of LUAD were performed by using the TISIDB database. Finally, the results of network pharmacology analysis were validated by qPCR. RESULTS: Experiments in vitro revealed that 6-MDS significantly reduced tumor growth. A total of 33 potential targets of 6-MDS in LUAD were obtained by crossing the LUAD related targets with 6-MDS targets. Utilizing CytoHubba, a network analysis tool, the top 10 genes with the highest centrality measures were pinpointed, including MMP9, CDK1, TYMS, CCNA2, ERBB2, CHEK1, KIF11, AURKB, PLK1 and TTK. Analysis of KEGG enrichment hinted that these 10 hub genes were located in the cell cycle signaling pathway, suggesting that 6-MDS may mainly inhibit the occurrence of LUAD by affecting the cell cycle. Molecular docking analysis revealed that the binding energies between 6-MDS and the hub proteins were all higher than - 6 kcal/Mol with the exception of AURKB, indicating that the 9 targets had strong binding ability with 6-MDS.These results were corroborated through assessments of mRNA expression levels, protein expression levels, overall survival analysis, promotor methylation level, immune subtypes andimmune infiltration. Furthermore, qPCR results indicated that 6-MDS can significantly decreased the mRNA levels of CDK1, CHEK1, KIF11, PLK1 and TTK. CONCLUSIONS: According to our findings, it appears that 6-MDS could possibly serve as a promising option for the treatment of LUAD. Further investigations in live animal models are necessary to confirm its potential in fighting cancer and to delve into the mechanisms at play.

Detection of HBV DNA integration in plasma cell-free DNA of different HBV diseases utilizing DNA capture strategy.

The landscape of hepatitis B virus (HBV) integration in the plasma cell-free DNA (cfDNA) of HBV-infected patients with different stages of liver diseases [chronic hepatitis B (CHB), liver cirrhosis (LC), and hepatocellular carcinoma (HCC)] remains unclear. In this study, we developed an improved strategy for detecting HBV DNA integration in plasma cfDNA, based on DNA probe capture and next-generation sequencing. Using this optimized strategy, we successfully detected HBV integration events in chimeric artificial DNA samples and HBV-infected HepG2-NTCP cells at day one post infection, with high sensitivity and accuracy. The characteristics of HBV integration events in the HBV-infected HepG2-NTCP cells and plasma cfDNA from HBV-infected individuals (CHB, LC, and HCC) were further investigated. A total of 112 and 333 integration breakpoints were detected in the HepG2-NTCP cells and 22 out of 25 (88%) clinical HBV-infected samples, respectively. In vivo analysis showed that the normalized number of support unique sequences (nnsus) in HCC was significantly higher than in CHB or LC patients (P values â€‹< â€‹0.05). All integration breakpoints are randomly distributed on human chromosomes and are enriched in the HBV genome around nt 1800. The majority of integration breakpoints (61.86%) are located in the gene-coding region. Both non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ) interactions occurred during HBV integration across the three different stages of liver diseases. Our study provides evidence that HBV DNA integration can be detected in the plasma cfDNA of HBV-infected patients, including those with CHB, LC, or HCC, using this optimized strategy.

A feature optimization study based on a diabetes risk questionnaire.

Introduction: The prevalence of diabetes, a common chronic disease, has shown a gradual increase, posing substantial burdens on both society and individuals. In order to enhance the effectiveness of diabetes risk prediction questionnaires, optimize the selection of characteristic variables, and raise awareness of diabetes risk among residents, this study utilizes survey data obtained from the risk factor monitoring system of the Centers for Disease Control and Prevention in the United States. Methods: Following univariate analysis and meticulous screening, a more refined dataset was constructed. This dataset underwent preprocessing steps, including data distribution standardization, the application of the Synthetic Minority Oversampling Technique (SMOTE) in combination with the Round function for equilibration, and data standardization. Subsequently, machine learning (ML) techniques were employed, utilizing enumerated feature variables to evaluate the strength of the correlation among diabetes risk factors. Results: The research findings effectively delineated the ranking of characteristic variables that significantly influence the risk of diabetes. Obesity emerges as the most impactful factor, overshadowing other risk factors. Additionally, psychological factors, advanced age, high cholesterol, high blood pressure, alcohol abuse, coronary heart disease or myocardial infarction, mobility difficulties, and low family income exhibit correlations with diabetes risk to varying degrees. Discussion: The experimental data in this study illustrate that, while maintaining comparable accuracy, optimization of questionnaire variables and the number of questions can significantly enhance efficiency for subsequent follow-up and precise diabetes prevention. Moreover, the research methods employed in this study offer valuable insights into studying the risk correlation of other diseases, while the research results contribute to heightened societal awareness of populations at elevated risk of diabetes.

Diabetes prediction model for unbalanced community follow-up data set based on optimal feature selection and scorecard.

Objectives: Diabetes is a metabolic disease and early detection is crucial to ensuring a healthy life for people with prediabetes. Community care plays an important role in public health, but the association between community follow-up of key life characteristics and diabetes risk remains unclear. Based on the method of optimal feature selection and risk scorecard, follow-up data of diabetes patients are modeled to assess diabetes risk. Methods: We conducted a study on the diabetes risk assessment model and risk scorecard using follow-up data from diabetes patients in Haizhu District, Guangzhou, from 2016 to 2023. The raw data underwent preprocessing and imbalance handling. Subsequently, features relevant to diabetes were selected and optimized to determine the optimal subset of features associated with community follow-up and diabetes risk. We established the diabetes risk assessment model. Furthermore, for a comprehensible and interpretable risk expression, the Weight of Evidence transformation method was applied to features. The transformed features were discretized using the quantile binning method to design the risk scorecard, mapping the model's output to five risk levels. Results: In constructing the diabetes risk assessment model, the Random Forest classifier achieved the highest accuracy. The risk scorecard obtained an accuracy of 85.16%, precision of 87.30%, recall of 80.26%, and an F1 score of 83.27% on the unbalanced research dataset. The performance loss compared to the diabetes risk assessment model was minimal, suggesting that the binning method used for constructing the diabetes risk scorecard is reasonable, with very low feature information loss. Conclusion: The methods provided in this article demonstrate effectiveness and reliability in the assessment of diabetes risk. The assessment model and scorecard can be directly applied to community doctors for large-scale risk identification and early warning and can also be used for individual self-examination to reduce risk factor levels.

Monomeric C-reactive protein is associated with severity and prognosis of decompensated hepatitis B cirrhosis.

C-reactive protein (CRP) is an acute-phase protein produced by the liver in response to infection and during chronic inflammatory disorders. Systemic inflammation is a major driver of cirrhosis progression from the compensated to the decompensated stage. Previous studies have shown that pentameric CRP (pCRP) to be a weak predictor of disease severity and prognosis in patients with decompensated hepatitis B cirrhosis, with it being only helpful for identifying patients with a higher short-term risk of death under certain conditions. Accumulating evidence indicates that pCRP dissociates to and acts primarily as the monomeric conformation (mCRP) at inflammatory loci, suggesting that mCRP may be a potentially superior disease marker with higher specificity and relevance to pathogenesis. However, it is unknown whether mCRP and anti-mCRP autoantibodies are associated with disease severity, or progression in decompensated hepatitis B cirrhosis. In this study, we evaluated the serum levels of mCRP and anti-mCRP autoantibodies in patients with decompensated cirrhosis of hepatitis B and their association with disease severity and theoretical prognosis. The results showed that patients with high mCRP and anti-mCRP autoantibody levels had more severe liver damage and that coagulation function was worse in patients with high anti-mCRP autoantibodies. Analysis of the correlation between pCRP, mCRP and anti-mCRP autoantibody levels with Model for End-Stage Liver Disease (MELD), Albumin-Bilirubin (ALBI), and Child-Turcotte-Pugh (CTP) prognostic scores showed that mCRP was the most strongly correlated with MELD score, followed by anti-mCRP autoantibodies; conversely, pCRP was not significantly correlated with prognostic score. Therefore, mCRP and anti-mCRP autoantibodies may be more advantageous clinical indicators than pCRP for evaluating the pathological state of decompensated hepatitis B cirrhosis.

Bioinformatics Prediction and Experimental Validation of the Role of Macrophage Polarization and Ferroptosis in Gestational Diabetes Mellitus.

Purpose: Gestational diabetes mellitus (GDM) is a common metabolic disorder during pregnancy that is associated with placental inflammation and adverse pregnancy outcomes. However, the mechanisms of inflammation in GDM are still unclear. Methods: Bulk transcriptome, single-cell transcriptome, clinical information, and samples were collected from GSE154414, GSE70493, GSE173193 and a retrospective cohort. Bioinformatics prediction was used to explore the mechanisms of placental inflammation, and multiplex immunofluorescence was used to validate the results. Results: First, we found that GDM is characterized by low-grade inflammation and is linked to several adverse pregnancy outcomes, as supported by our collected clinical data. Additionally, we identified ten hub genes (FCGR3B, CXCR1, MMP9, ITGAX, CCL5, GZMB, S100A8, LCN2, TGFB1, and LTF) as potential therapy targets and confirmed the binding of corresponding predictive therapeutic agents by molecular docking. Transcriptome sequencing analysis has shown that macrophages are primarily responsible for the emergence of placental inflammation, and that M1 macrophage polarization increased while M2 macrophage polarization decreased in GDM when compared to the control sample. Multiplex immunofluorescence staining of CD68, CD80, and ACSL4 was performed and suggested that ferroptosis of macrophages may contribute to placental inflammation in GDM. Conclusion: In conclusion, our findings provide a better understanding of the mechanisms of inflammation in GDM and suggest potential therapeutic targets for this condition.

Identification and Validation a Necroptosis-Related Prognostic Signature in Cervical Cancer.

Necroptosis is a promising novel target for cervical cancer therapy. Nevertheless, differentially expressed necroptosis-related genes (NRGs) in cervical cancer and their associations with prognosis are far from fully clarified. In this study, differentially expressed NRGs (DE-NRGs) were screened out and their bio-function was elucidated. Subsequently, a prognostic scoring model based on the regression coefficients of the screened out NRGs and their corresponding mRNA expressions were constructed and validated. Finally, the survival probability of cervical cancer patients based on the constructed prognostic scoring model in 3 and 5 years was predicted and assessed. We found 17 DE-NRGs in cervical cancer tissues which were closely related to cancer progression, and most of them were significantly highly expressed. Furthermore, 3 NRG were confirmed as the prognostic signature genes from 17 DE-NRGs by regression analysis. Overall survival predicted through our prognostic scoring model was lower in the high-risk group than in the low-risk group (p < 0.05) in both the TCGA cohort and the external GEO44001 validation cohort. What's more, the prediction performance of our prognostic scoring models well verified by the ROC curve, and the risk score calculated could act as an independent prognostic factor for cervical cancer patients. The calibration curve and C-index (0.776) of the nomogram analysis suggested that the predictive performance of the nomogram was satisfactory. Our study identified and validated a necroptosis-related prognostic signature in cervical cancer, which could well predict the prognosis for cervical cancer patients.

Genome-wide analyzation and functional characterization on the TPS family provide insight into the biosynthesis of mono-terpenes in the camphor tree.

Terpene synthase (TPS) plays an important role in terpenoids biosynthesis. Cinnamomum camphora (camphor tree) contains dozens of terpenoids with medicinal value, especially borneol, which has been widely used since ancient times. However, limited information is available regarding the genome-wide identification and characterization of the TPS family in the C. camphora. In this study, 82 CcTPS genes were identified from the camphor tree genome (CTG). Gene cluster and sequence syntenic analysis suggested that tandem duplication occurred within the TPS family of the CTG, especially for the TPS-b subfamily. The chemotype-specific gene expression analysis showed significantly differential expression patterns among six chemotypes. It is worth noting that three genes (CcTPS26, CcTPS49 and CcTPS72) exhibited relatively high expression in the borneol-type camphor tree, compared to the other five chemotypes. Further functional characterization of them indicated that they were all bornyl diphosphate synthases (BPPSs), which function in catalyzing GPP into BPP and then undergoes dephosphorylation to yield borneol. This is the first report that multiple BPPSs exist within a single species. Intriguingly, CcTPS49 and CcTPS72 lead to the generation of dextral-borneol, while CcTPS26 contributes to the biosynthesis of levo-borneol. In addition, the functional characterization of another six CcTPSs suggested that they are responsible for the biosynthesis of linalool, eucalyptol and several other monoterpenes in camphor tree. In conclusion, these novel results provide a foundation for further exploration of the role of the CcTPS gene family and shed light on a better understanding of the biosynthesis and accumulation of monoterpenes in camphor tree.

The association between urinary organophosphate insecticide metabolites and erectile dysfunction in the United States.

Organophosphate (OP) insecticides are the main chemicals used in agriculture for pest elimination, and they have been linked with many diseases. However, there is no literature regarding the impacts of organophosphate insecticide metabolite exposure on erectile dysfunction (ED). We aimed to evaluate the correlation between 4 urinary organophosphate insecticide metabolites and the presence of ED in a representative sample of men aged 20 and older. The dataset including a total of 555 subjects was obtained from the National Health and Nutrition Examination Survey (NHANES) 2003-2004. ED was assessed by a question from a self-report questionnaire. Weighted proportions and multivariable logistic regression analysis were utilized to examine the relationship between organophosphate insecticide metabolite exposure and ED. In multivariable logistic regression analysis, diethylphosphate (DEP) was positively correlated with ED (OR 1.07; 95% CI 1.01-1.14; P = 0.033) after full adjustment. Men in DEP tertile 4 had a significant 33% higher risk of ED than those in tertile 1. Furthermore, in a subgroup analysis, our results showed that higher DEP levels were significantly associated with ED in the young age group (20 ≤ age ≤ 39). Our study revealed a significant association between organophosphate insecticide metabolite exposure and an increased risk of ED. Moreover, the correlations were more evident in the young age group. The evaluation of urinary organophosphate insecticide metabolite exposure should be included in the risk assessment of ED. Further study to investigate the underlying mechanism, such as how long the urinary metabolite is present, whether ED is reversible in this population by lowering DEP concentrations, and how exposure to this metabolite affects erectile tissue, is warranted.

Characterization of the Complete Chloroplast Genome of Buddleja lindleyana.

BACKGROUND: Buddleja lindleyana Fort., which belongs to the Loganiaceae with a distribution throughout the tropics, is widely used as an ornamental plant in China. There are several morphologically similar species in the genus Buddleja, but the lack of comprehensive molecular and phylogenetic studies makes it difficult to distinguish related species, which hinders further studies of this genus. OBJECTIVE: Using molecular biology techniques to sequence and analyze the complete chloroplast (cp) genome of B. lindleyana. METHODS: After sequencing of the genomic DNA using next-generation sequencing, a series of bioinformatics software were used to assemble and analyze the molecular structure of the cp genome of B. lindleyana. RESULTS: The complete cp genome of B. lindleyana is a circular 154 487-bp-long molecule with a GC (Guanine and Cytosine) content of 38.1%. It has a quadripartite structure, including a LSC region (85 489 base pair (bp)), a small single-copy region (17 898 bp), and a pair of inverted repeat regions (25 550 bp). A total of 133 genes were identified in this genome, including 86 protein-coding genes, 37 tRNA (transfer Ribonucleic Acid) genes, eight rRNA (ribosomal Ribonucleic Acid) genes, and two pseudogenes. CONCLUSION: These results suggest that the B. lindelyana cp genome could be used as a potential genomic resource to resolve the phylogenetic positions and relationships of Loganiaceae, and will offer valuable information for future research in the identification of Buddleja species and will conduce to genomic investigations into these species. HIGHLIGHTS: This paper study the B. lindelyana cp genome and it's structural characteristics, and analyze the phylogeny of Loganiaceae.

Comparative genomic study on the complete plastomes of four officinal Ardisia species in China.

Ardisia Sw. (Primulaceae) is naturally distributed in tropical and subtropical areas. Most of them possess edible and medicinal values and are popular in clinical and daily use in China. However, ambiguous species delineation and genetic information limit the development and utilization of this genus. In this study, the chloroplast genomes of four Ardisia species, namely A. gigantifolia Stapf, A. crenata Sims, A. villosa Roxb. and A. mamillata Hance, were sequenced, annotated, and analyzed comparatively. All the four chloroplast genomes possess a typical quadripartite structure, and each of the genomes is about 156 Kb in size. The structure and gene content of the Ardisia plastomes were conservative and showed low sequence divergence. Furthermore, we identified five mutation hotspots as candidate DNA barcodes for Ardisia, namely, trnT-psbD, ndhF-rpl32, rpl32-ccsA, ccsA-ndhD and ycf1. Phylogenetic analysis based on the whole-chloroplast genomes data showed that Ardisia was sister to Tapeinosperma Hook. f. In addition, the results revealed a great topological profile of Ardisia's with strong support values, which matches their geographical distribution patterns. Summarily, our results provide useful information for investigations on taxonomic differences, molecular identification, and phylogenetic relationships of Ardisia plants.

Metabolism and transcriptome profiling provides insight into the genes and transcription factors involved in monoterpene biosynthesis of borneol chemotype of Cinnamomum camphora induced by mechanical damage.

BACKGROUND: The borneol chemotype of Cinnamomum camphora (BCC), a monoterpene-rich woody plant species, is the sole source prescribed by the Chinese Pharmacopoeia for the production of natural D-borneol, a major monoterpene in BCC used for millennia as a topical analgesic in China. Nevertheless, the possible gene-regulatory roles of transcription factors (TFs) in BCC's monoterpenoid biosynthesis remained unknown. Here, a joint analysis of the transcriptome and terpenoid metabolome of BCC induced by mechanical damage (MD) was used to comprehensively explore the interaction between TFs and terpene synthase (TPS) unigenes that might participate in monoterpene biosynthesis in BCC. RESULTS: Gas chromatography-mass spectrometry analysis detected 14 monoterpenes and seven sesquiterpenes. All but two monoterpenes underwent a significantly increased accumulation after the MD treatment. RNA sequencing data revealed that 10 TPS, 82 MYB, 70 AP2/ERF, 38 BHLH, 31 WRKY, and 29 bZIP unigenes responded to the MD treatment. A correlation analysis revealed that three monoterpene synthase genes (CcTPS1, CcTPS3, CcTPS4) highly correlated with multiple monoterpenes, namely D-borneol, camphor, and bornyl acetate, which could be responsible for monoterpenoid biosynthesis in BCC. Furthermore, five WRKY, 15 MYB, 10 ERF/AP2, five bZIP, and two BHLH genes had strong, positive correlations with CcTPS1 or CcTPS4, judging by their high coefficient values (R2 > 0.8). The bioinformatics results were verified by quantitative real-time PCR. CONCLUSION: This study provides insight into the genes involved in the biosynthesis and regulation of monoterpene in BCC and thus provides a pool of candidate genes for future mechanistic analyses of how monoterpenes accumulate in BCC.

Genome-Wide Identification and Functional Characterization of the Trans-Isopentenyl Diphosphate Synthases Gene Family in Cinnamomum camphora.

Trans-isopentenyl diphosphate synthases (TIDSs) genes are known to be important determinants for terpene diversity and the accumulation of terpenoids. The essential oil of Cinnamomum camphora, which is rich in monoterpenes, sesquiterpenes, and other aromatic compounds, has a wide range of pharmacological activities and has therefore attracted considerable interest. However, the TIDS gene family, and its relationship to the camphor tree (C. camphora L. Presl.), has not yet been characterized. In this study, we identified 10 TIDS genes in the genome of the C. camphora borneol chemotype that were unevenly distributed on chromosomes. Synteny analysis revealed that the TIDS gene family in this species likely expanded through segmental duplication events. Furthermore, cis-element analyses demonstrated that C. camphora TIDS (CcTIDS) genes can respond to multiple abiotic stresses. Finally, functional characterization of eight putative short-chain TIDS proteins revealed that CcTIDS3 and CcTIDS9 exhibit farnesyl diphosphate synthase (FPPS) activity, while CcTIDS1 and CcTIDS2 encode geranylgeranyl diphosphate synthases (GGPPS). Although, CcTIDS8 and CcTIDS10 were found to be catalytically inactive alone, they were able to bind to each other to form a heterodimeric functional geranyl diphosphate synthase (GPPS) in vitro, and this interaction was confirmed using a yeast two-hybrid assay. Furthermore, transcriptome analysis revealed that the CcTIDS3, CcTIDS8, CcTIDS9, and CcTIDS10 genes were found to be more active in C. camphora roots as compared to stems and leaves, which were verified by quantitative real-time PCR (qRT-PCR). These novel results provide a foundation for further exploration of the role of the TIDS gene family in camphor trees, and also provide a potential mechanism by which the production of camphor tree essential oil could be increased for pharmacological purposes through metabolic engineering.

Effects of Different Molecular Weight Polysaccharides From Dendrobium officinale Kimura & Migo on Human Colorectal Cancer and Transcriptome Analysis of Differentially Expressed Genes.

We investigated the antitumor effects of four fractions of Dendrobium officinale Kimura & Migo (D. officinale) polysaccharides with different molecular weights (Mw), Astragalus membranaceus polysaccharides (APS) and Lentinus edodes polysaccharides (LNT) on colorectal cancer (CRC) using a zebrafish xenograft model. Transcriptome sequencing was performed to further explore the possible antitumor mechanisms of D. officinale polysaccharides. Fractions of D. officinale polysaccharides, LNT, and APS could significantly inhibit the growth of HT-29 cells in a zebrafish xenograft model. One fraction of D. officinale polysaccharides called DOPW-1 (Mw of 389.98 kDa) exhibited the strongest tumor inhibition. Compared with the control group, RNA-seq revealed that the DOPW-1-treated experimental group had 119 differentially expressed genes (DEGs), of which 45 had upregulated expression and 74 had downregulated expression. Analyses using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes suggested that the pathway "apoptosis-multiple species" was the most significantly enriched. Our data indicated that 1) fractions of D. officinale polysaccharides of Mw 389.98 kDa were most suitable against CRC; 2) DOPW-1 could be developed into a clinical agent against CRC; and 3) an apoptosis pathway is important for DOPW-1 to inhibit the proliferation of HT-29 cells.

Mining of candidate genes involved in the biosynthesis of dextrorotatory borneol in Cinnamomum burmannii by transcriptomic analysis on three chemotypes.

BACKGROUND: Dextrorotatory borneol (D-borneol), a cyclic monoterpene, is widely used in traditional Chinese medicine as an efficient topical analgesic drug. Fresh leaves of Cinnamomum trees, e.g., C. burmannii and C. camphor, are the main sources from which D-borneol is extracted by steam distillation, yet with low yields. Insufficient supply of D-borneol has hampered its clinical use and production of patent remedies for a long time. Biological synthesis of D-borneol offers an additional approach; however, mechanisms of D-borneol biosynthesis remain mostly unresolved. Hence, it is important and necessary to elucidate the biosynthetic pathway of D-borneol. RESULTS: Comparative analysis on the gene expression patterns of different D-borneol production C. burmannii samples facilitates elucidation on the underlying biosynthetic pathway of D-borneol. Herein, we collected three different chemotypes of C. burmannii, which harbor different contents of D-borneol.A total of 100,218 unigenes with an N50 of 1,128 bp were assembled de novo using Trinity from a total of 21.21 Gb clean bases. We used BLASTx analysis against several public databases to annotate 45,485 unigenes (45.38%) to at least one database, among which 82 unigenes were assigned to terpenoid biosynthesis pathways by KEGG annotation. In addition, we defined 8,860 unigenes as differentially expressed genes (DEGs), among which 13 DEGs were associated with terpenoid biosynthesis pathways. One 1-deoxy-D-xylulose-5-phosphate synthase (DXS) and two monoterpene synthase, designated as CbDXS9, CbTPS2 and CbTPS3, were up-regulated in the high-borneol group compared to the low-borneol and borneol-free groups, and might be vital to biosynthesis of D-borneol in C. burmannii. In addition, we identified one WRKY, two BHLH, one AP2/ERF and three MYB candidate genes, which exhibited the same expression patterns as CbTPS2 and CbTPS3, suggesting that these transcription factors might potentially regulate D-borneol biosynthesis. Finally, quantitative real-time PCR was conducted to detect the actual expression level of those candidate genes related to the D-borneol biosynthesis pathway, and the result showed that the expression patterns of the candidate genes related to D-borneol biosynthesis were basically consistent with those revealed by transcriptome analysis. CONCLUSIONS: We used transcriptome sequencing to analyze three different chemotypes of C. burmannii, identifying three candidate structural genes (one DXS, two monoterpene synthases) and seven potential transcription factor candidates (one WRKY, two BHLH, one AP2/ERF and three MYB) involved in D-borneol biosynthesis. These results provide new insight into our understanding of the production and accumulation of D-borneol in C. burmannii.