Identification of P450 Candidates Associated with the Biosynthesis of Physalin-Class Compounds in Physalis angulata.

The Physalis genus has long been used as traditional medicine in the treatment of various diseases. Physalins, the characteristic class of compounds in this genus, are major bioactive constituents. To date, the biogenesis of physalins remains largely unknown, except for the recently established knowledge that 24-methyldesmosterol is a precursor of physalin. To identify the genes encoding P450s that are putatively involved in converting 24-methyldesmosterol to physalins, a total of 306 P450-encoding unigenes were retrieved from our recently constructed P. angulata transcriptome. Extensive phylogenetic analysis proposed 21 P450s that might participate in physalin biosynthesis. To validate the candidates, we developed a virus-induced gene silencing (VIGS) system for P. angulata, and four P450 candidates were selected for the VIGS experiments. The reduction in the transcripts of the four P450 candidates by VIGS all led to decreased levels of physalin-class compounds in the P. angulata leaves. Thus, this study provides a number of P450 candidates that are likely associated with the biosynthesis of physalin-class compounds, forming a strong basis to reveal the unknown physalin biosynthetic pathway in the future.

Serum metabolomics analysis of biomarkers and metabolic pathways in patients with colorectal cancer associated with spleen-deficiency and qi-stagnation syndrome or damp-heat syndrome: a prospective cohort study.

Objective: To profile the serum metabolites and metabolic pathways in colorectal cancer (CRC) patients associated with spleen-deficiency and qi-stagnation syndrome (SDQSS) or damp-heat syndrome (DHS). Methods: From May 2020 to January 2021, CRC patients diagnosed with traditional Chinese medicine (TCM) syndromes of SDQSS or DHS were enrolled. The clinicopathological data of the SDQSS and DHS groups were compared. The serum samples were analyzed by liquid chromatography-mass spectrometry (LC-MS). The variable importance in the projection >1, fold change ≥3 or ≤0.333, and P value ≤0.05 were used to identify differential metabolites between the two groups. Furthermore, areas under the receiver operating characteristic (ROC) curve > 0.9 were applied to select biomarkers with good predictive performance. The enrichment metabolic pathways were searched through the database of Kyoto Encyclopedia of Genes and Genomes. Results: 60 CRC patients were included (30 SDQSS and 30 DHS). The level of alanine aminotransferase was marginally significantly higher in the DHS group than the SDQSS group (P = 0.051). The other baseline clinicopathological characteristics were all comparable between the two groups. 23 differential serum metabolites were identified, among which 16 were significantly up-regulated and 7 were significantly down-regulated in the SDQSS group compared with the DHS group. ROC curve analysis showed that (S)-3-methyl-2-oxopentanoic acid, neocembrene, 1-aminocyclopropanecarboxylic acid, 3-methyl-3-hydroxypentanedioate, and nicotine were symbolic differential metabolites with higher predictive power. The top five enrichment signalling pathways were valine, leucine and isoleucine biosynthesis; lysosome; nicotine addiction; fructose and mannose metabolism; and pertussis. Conclusion: Our study identifies the differential metabolites and characteristic metabolic pathways among CRC patients with SDQSS or DHS, offering the possibility of accurate and objective syndrome differentiation and TCM treatment for CRC patients.

Glycosylation and methylation in the biosynthesis of isoflavonoids in Pueraria lobata.

The pathway for forming isoflavonoid skeletal structure is primarily restricted to the Leguminosae family. Subsequent decorations on the compound backbone by tailoring enzymes would change their biological and medicinal properties. Pueraria lobata is a leguminous plant, and as a traditional Chinese medicine its roots have been ascribed a number of pharmacological activities. Glycosylation and methylation are the main modifying processes in isoflavonoid metabolism in P. lobata roots, resulting in the accumulation of unique glycosylated and methylated end isoflavonoid compounds. For instance, daidzein 8-C-glucoside (i.e., puerarin) and puerarin derivatives are produced only by the Pueraria genus. Puerarin has been established as a clinical drug for curing cardiovascular diseases. To better understand the characteristic isoflavonoid metabolism in P. lobata, this review attempts to summarize the research progress made with understanding the main glycosylation and methylation of isoflavonoids in P. lobata and their biosynthetic enzymes.

RNAi of Sterol Δ24-Isomerase Implicated Its Involvement in Physalin Biosynthesis in Physalis angulata L.

Physalis angulata is a renowned traditional Chinese medicine for the treatment of various conditions. Physalin is the major type of bioactive constituents conferring medicinal properties of P. angulata. Despite the medicinal importance, the pathways leading to physalin are largely unknown. In this study, we employed a transcriptomic approach to identify a Pa24ISO gene from P. angulata. Through heterologous expression in yeast, Pa24ISO was revealed to catalyze an isomerization reaction in converting 24-methylenecholesterol to 24-methyldesmosterol. Real-time PCR analysis showed that the abundance of Pa24ISO transcripts correlated with the accumulation pattern of physalin B in different tissues of P. angulata. A direct role of Pa24ISO in channeling of 24-methylenecholesterol for physalin B biosynthesis was illustrated by suppressing the gene in P. angulata via the VIGS approach. Down-regulation of Pa24ISO led to reduced levels of 24-methyldesmosterol and physalin B, accompanied with an increase of campesterol content in P. angulata. The results supported that 24ISO is involved in physalin biosynthesis in plants.

A Composite Deferoxamine/Black Phosphorus Nanosheet/Gelatin Hydrogel Scaffold for Ischemic Tibial Bone Repair.

Introduction: Bone delay union is mostly caused by lack of blood supply. Although autografts, allografts and artificial bone have been widely used to treat bone delay union, the bone regeneration fails in the ischemic site accompanied by the bone donor site complications and disease transmission. Recently, there is a growing recognition of the importance of hydrogel scaffolds which are regarded as an eligible engineer tissue for bone repair. However, hydrogel is still limited in improving neovascularization. Methods: In this work, black phosphorus nanosheet and deferoxamine (BPN-DFO) were loaded in the gelatin hydrogel to overcome the high risk of bone delay union and systemically investigated the regeneration capability of BPN-DFO hydrogel in vitro and vivo. Results: The resulting BPN-DFO hydrogel scaffold showed superior swollen, degradation and release rate, as well as satisfied biocompatibility. BPN-DFO hydrogel shown the significant up-expression of mRNA related to bone regeneration and cell proliferation. In vivo, the proposed BPN-DFO hydrogel significantly improved osteogenesis and neovascularization in the ischemic tibial bone site of SD rats with acute femoral artery occlusion. Both macroscopic and histological evaluation of new regenerated bone showed newly formed blood vessel and collagen using BPN-DFO hydrogel. The immunohistochemistry and RT-PCR revealed that the bone regeneration could be improved via BMP/Runx2 pathway. Conclusion: The BPN-DFO hydrogel possesses potential tissue engineer material for ischemic bone defect treatment. However, furthermore studies are needed to testify the safety and efficacy of BPN-DFO hydrogel.

Antimicrobial Lignin-Based Polyurethane/Ag Composite Foams for Improving Wound Healing.

Antimicrobial materials are an urgent need for modern wound care in the clinic. Although traditional polyurethane foams have proven to be clinically valuable for wound treatment, their petroleum-originated preparation and bioinert nature have restricted their efficacy in biomedical applications. Here, we propose a simple one-step foaming method to prepare lignin-based polyurethane foams (LPUFs) in which fully biobased polyether polyols partially replace traditional petroleum-based raw materials. The trace amount of phenolic hydroxyl groups (about 4 mmol) in liquefied lignin acts as a direct reducing agent and capping agent to silver ions (less than 0.3 mmol), in situ forming silver nanoparticles (Ag NPs) within the LPUF skeleton. This newly proposed lignin polyurethane/Ag composite foam (named as Ag NP-LPUF) shows improved mechanical, thermal, and antibacterial properties. It is worth mentioning that the Ag NP-LPUF exhibits more than 99% antibacterial rate against Escherichia coli within 1 h and Staphylococcus aureus within 4 h. Evaluations in mice indicate that the antimicrobial composite foams can effectively promote wound healing of full-thickness skin defects. As a proof of concept, this antibacterial and biodegradable foam exhibits significant potential for clinical translation in wound care dressings.

Chromosomal-level genome and multi-omics dataset of Pueraria lobata var. thomsonii provide new insights into legume family and the isoflavone and puerarin biosynthesis pathways.

Pueraria lobata var. thomsonii (hereinafter abbreviated as Podalirius thomsonii), a member of legumes, is one of the important traditional Chinese herbal medicines, and its puerarin extraction is widely used in health and pharmaceutical industry. Here, we assembled a high-quality genome of P. thomsonii using long-read single-molecule sequencing and Hi-C technologies. The genome assembly is approximately 1.37 Gb in size and consists of 5145 contigs with a contig N50 of 593.70 Kb, further clustered into 11 pseudochromosomes. The genome structural annotation resulted in about 869.33 Mb (about 62.70% of the genome) repeat regions and 45 270 protein-coding genes. Genome evolution analysis revealed that P. thomsonii is most closely related to soybean and underwent two ancient whole-genome duplication events, one was in the common ancestor shared by legume species, the other occurred independently at around 7.2 million years ago after its specification. A total of 2373 gene families were found unique in P. thomsonii comparing to five other legume species. Genes and metabolites related to puerarin content in tuberous tissues were characterized. A total of 572 genes upregulated in the puerarin biosynthesis pathway were identified, and 235 candidate genes were further enriched by omics data. Furthermore, we identified 6 8-C-glucosyltransferase (8-C-GT) candidate genes significantly involved in puerarin metabolism. Our study filled in a key genomic gap in legume family, and provided valuable multi-omic resources for the genetic improvement of P. thomsonii.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Feedback of biochemical indexes of blood sugar control by swimming exercise / Feedback dos índices bioquímicos de controle de açúcar no sangue por exercícios de natação / Feedback de los índices bioquímicos del control del azúcar en sangre mediante el ejercicio de natación

ABSTRACT Introduction: According to the metabolic characteristics of ultra-long-distance swimming and the characteristics of energy utilization and absorption during exercise, we have formulated a nutritional supplement plan for crossing to study the influence of swimming sports on blood sugar and give biochemical feedback indicators. Objective: To lay a foundation for studying the nutrition supplement rules during long-term exercise by taking the athletes' blood after training to determine the changes in blood sugar, adjusting and determining the nutritional supplement plan during training. Methods: We monitor athletes' physical function changes and biochemical indicators during training and study the changes of these biochemical indicators and athletes' physical functions after long-term swimming exercises to scientifically arrange the exercise intensity and load during the training period. Results: The urine indexes after exercise did not change much, reflecting the exercise load's low intensity. The changes in blood lactic acid and blood urea indexes after exercise also confirmed this. During the training period, the athletes' hemoglobin and red blood cell parameters are in the ideal range, indicating that the athlete's physical function is in good condition. During the training period, the training load intensity and load are reasonable according to ultra-long-distance swimming's energy supply characteristics. After training, the changes in blood glucose indicators reflect that the nutritional supplement program we formulated for athletes is reasonable and feasible. Conclusions: By monitoring the blood sugar and biochemical indicators of swimmers, it can help athletes to arrange exercise intensity scientifically and load during the training period, to better carry out open water competitions in China, and to arrange training and scientific nutrition during the training period scientifically. Lay the foundation for the establishment of nutrition supplement theory and training theory for super long-time sports. Level of evidence II; Therapeutic studies - investigation of treatment results.

RESUMEN Introducción: De acuerdo con las características metabólicas de la natación de ultra larga distancia y las características de utilización y absorción de energía durante el ejercicio, hemos formulado un plan de complementos nutricionales para estudiar la influencia de los deportes de natación en el azúcar en sangre y dar indicadores de retroalimentación bioquímica. Objetivo: Sentar las bases para el estudio de las reglas de los suplementos nutricionales durante el ejercicio a largo plazo mediante la extracción de sangre de los atletas después del entrenamiento para determinar los cambios en el azúcar en sangre, ajustando y determinando el plan de suplementos nutricionales durante el entrenamiento. Métodos: monitoreamos los cambios en la función física de los atletas y los indicadores bioquímicos durante el entrenamiento y estudiamos los cambios de estos indicadores bioquímicos y las funciones físicas de los atletas después de ejercicios de natación de larga distancia para organizar científicamente la intensidad y la carga del ejercicio durante el período de entrenamiento. Resultados: Los índices de orina después del ejercicio no cambiaron mucho, lo que refleja la baja intensidad de la carga de ejercicio. Los cambios en los índices de ácido láctico y urea en sangre después del ejercicio también lo confirmaron. Durante el período de entrenamiento, los parámetros de hemoglobina y glóbulos rojos de los atletas están en el rango ideal, lo que indica que la función física del atleta está en buenas condiciones. Durante el período de entrenamiento, la intensidad de la carga de entrenamiento y la carga son razonables de acuerdo con las características de suministro de energía de la natación de ultra larga distancia. Después del entrenamiento, los cambios en los indicadores de glucosa en sangre reflejan que el programa de suplementos nutricionales que formulamos para los atletas es razonable y factible. Conclusiones: monitorear los indicadores bioquímicos y de azúcar en sangre de los nadadores, puede ayudar a los atletas a organizar científicamente la intensidad del ejercicio y la carga durante el período de entrenamiento, a realizar mejor las competiciones en aguas abiertas en China y a organizar el entrenamiento y la nutrición científica durante el período de entrenamiento. Sentar las bases para el establecimiento de la teoría de los suplementos nutricionales y la teoría del entrenamiento para deportes de larga duración. Nivel de evidencia II; Estudios terapéuticos: investigación de los resultados del tratamiento.

RESUMO Introdução: De acordo com as características metabólicas da natação de ultra longa distância e as características de utilização e absorção de energia durante o exercício, formulamos um plano de suplemento nutricional para estudar a influência dos esportes de natação no açúcar no sangue e fornecer indicadores de feedback bioquímico. Objetivo: Estabelecer as bases para o estudo das regras de suplementos nutricionais durante exercícios de longa duração, retirando sangue de atletas após o treinamento para determinar as mudanças na glicemia, ajustando e determinando o plano de suplementação nutricional durante o treinamento. Métodos: monitoramos as mudanças na função física e nos indicadores bioquímicos dos atletas durante o treinamento e estudamos as mudanças nesses indicadores bioquímicos e nas funções físicas dos atletas após exercícios de natação de longa distância para organizar cientificamente a intensidade e a carga do exercício durante o período de treinamento. Resultados: As taxas de urina após o exercício não mudaram muito, refletindo a baixa intensidade da carga de exercício. Alterações nos índices de uréia e ácido láctico no sangue após o exercício também confirmaram isso. Durante o período de treinamento, os parâmetros de hemoglobina e hemácias dos atletas estão na faixa ideal, indicando que a função física do atleta está em boas condições. Durante o período de treinamento, a intensidade da carga de treinamento e a carga são razoáveis de acordo com as características da fonte de alimentação da natação de ultra longa distância. Após o treinamento, as mudanças nos indicadores de glicose no sangue refletem que o programa de suplementos nutricionais que formulamos para atletas é razoável e viável. Conclusões: monitorar os indicadores bioquímicos e de açúcar no sangue de nadadores pode ajudar os atletas a organizar cientificamente a intensidade e carga do exercício durante o período de treinamento, conduzir melhor competições em águas abertas na China e organizar treinamento e nutrição científica durante o período de treinamento. Estabelecendo as bases para o estabelecimento da teoria dos suplementos nutricionais e da teoria do treinamento para esportes de longa duração. Nível de evidência II; Estudos terapêuticos: investigação dos resultados do tratamento.

A Systematic Review of Traditionally Used Herbs and Animal-Derived Products as Potential Analgesics.

Pain is a distressing but fundamental manifestation that prepares the body for potentially detrimental stimuli while ensuring its protection. Plant and animal products have traditionally been used to relieve pain for centuries. However, no attempt has been made to compile a single report of plant and animal products possessing analgesic properties. This review enadeavours to recover data from published articles to establish a collective literature review on folk remedies from plant and animal sources used as analgesics and in the treatment of pain-related conditions, identifying gaps in existing knowledge and future works. Relevant information was systematically retrieved using the PRISMA method. In this review, in total, 209 plants were found to be either used raw or prepared by decoctions or maceration. Administration was either oral or topical, and they were predominantly used in Asian countries. In vivo studies of plants with analgesic properties, which were tested using different methods including acetic-induced writhing test, hotplate test, tail-flick test, and formalin-induced pain test, were compiled. Animal products with analgesic properties were obtained mainly from compounds present in venom; their bioactive compounds were also identified. In the literature search, certain gaps were noted, which could be reviewed in future studies. For instance, there was a disparity of information regarding the traditional uses of medicinal plants. In this review, an attempt was made to critically assess and describe the pharmacological properties and bioactive composition of indigenous plants, some animal species, and animal venom by scrutinizing databases and looking for published articles. Therefore, it can be concluded that the compounds obtained from these sources can serve as important ingredients in therapeutic agents to alleviate pain once their limitations are assessed and improved upon. In the literature search, certain gaps were noted, which could be reviewed in future studies.

Identification of Three (Iso)flavonoid Glucosyltransferases From Pueraria lobata.

(Iso)flavonoids are one of the largest groups of natural phenolic products conferring great value to the health of plants and humans. Pueraria lobata, a legume, has long been used in Chinese traditional medicine. (Iso)flavonoids mainly present as glycosyl-conjugates and accumulate in P. lobata roots. However, the molecular mechanism underlying the glycosylation processes in (iso)flavonoid biosynthesis are not fully understood. In the current study, three novel UDP-glycosyltransferases (PlUGT4, PlUGT15, and PlUGT57) were identified in P. lobata from RNA-seq data. Biochemical assays of these three recombinant PlUGTs showed all of them were able to glycosylate isoflavones (genistein and daidzein) at the 7-hydroxyl position in vitro. In comparison with the strict substrate specificity for PlUGT15 and PlUGT57, PlUGT4 displayed utilization of a broad range of sugar acceptors. Particularly, PlUGT15 exhibited a much higher catalytic efficiency toward isoflavones (genistein and daidzein) than any other identified 7-O-UGT from P. lobata. Moreover, the transcriptional expression patterns of these PlUGTs correlated with the accumulation of isoflavone glucosides in MeJA-treated P. lobata, suggesting their possible in vivo roles in the glycosylation process.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Discovery of a non-stereoselective cytochrome P450 catalyzing either 8α- or 8ß-hydroxylation of germacrene A acid from the Chinese medicinal plant, Inula hupehensis.

Sesquiterpene lactones (STLs) are C15 terpenoid natural products with α-methylene γ-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid (GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (α- or ß-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6α-, 12,6ß-, 12,8α-, and 12,8ß-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8α and C12,8ß) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 (CYP71BL6) that can catalyze the hydroxylation of GAA in either 8α- or 8ß-configuration, resulting in the synthesis of both 8α- and 8ß-hydroxyl GAAs. Of these two products, only 8α-hydroxyl GAA spontaneously lactonizes to the C12,8α-STL while the 8ß-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8α-STL, named inunolide, and 8ß-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71BL6 displays 63-66% amino acid identity to the previously reported CYP71BL1/2 catalyzing GAA 6α- or 8ß-hydroxylation, indicating CYP71BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71BL6 transcript abundance correlates closely to the accumulation of C12,8-STLs in I. hupehensis. The identification of CYP71BL6 provides an insight into the biosynthesis of STLs in Asteraceae.

Identification and Functional Characterization of Sesquiterpene Synthases from Xanthium strumarium.

Xanthium strumarium synthesizes various pharmacologically active sesquiterpenes. The molecular characterization of sesquiterpene biosynthesis in X. strumarium has not been reported so far. In this study, the cDNAs coding for three sesquiterpene synthases (designated as XsTPS1, XsTPS2 and XsTPS3) were isolated using the X. strumarium transcriptome that we recently constructed. XsTPS1, XsTPS2 and XsTPS3 were revealed to have primary activities forming germacrene D, guaia-4,6-diene and germacrene A, respectively, by either ectopic expression in yeast cells or purified recombinant protein-based in vitro assays. Quantitative real-time PCRs and metabolite analysis for the different plant parts showed that the transcript abundance of XsTPS1-XsTPS3 is consistent with the accumulation pattern of their enzymatic products, supporting their biochemical functions in vivo. In particular, we discovered that none of the XsTPS2 product, guaia-4,6-diene, can be detected in one of the X. strumarium cultivars used in this study (it was named the Hubei-cultivar), in which a natural deletion of two A bases in the XsTPS2 cDNA disrupts its activity, which further confirmed the proposed biochemical role of XsTPS2 in X. strumarium in vivo.

Differential microRNA Analysis of Glandular Trichomes and Young Leaves in Xanthium strumarium L. Reveals Their Putative Roles in Regulating Terpenoid Biosynthesis.

The medicinal plant Xanthium strumarium L. (X. strumarium) is covered with glandular trichomes, which are the sites for synthesizing pharmacologically active terpenoids such as xanthatin. MicroRNAs (miRNAs) are a class of 21-24 nucleotide (nt) non-coding RNAs, most of which are identified as regulators of plant growth development. Identification of miRNAs involved in the biosynthesis of plant secondary metabolites remains limited. In this study, high-throughput Illumina sequencing, combined with target gene prediction, was performed to discover novel and conserved miRNAs with potential roles in regulating terpenoid biosynthesis in X. strumarium glandular trichomes. Two small RNA libraries from leaves and glandular trichomes of X. strumarium were established. In total, 1,185 conserved miRNAs and 37 novel miRNAs were identified, with 494 conserved miRNAs and 18 novel miRNAs being differentially expressed between the two tissue sources. Based on the X. strumarium transcriptome data that we recently constructed, 3,307 annotated mRNA transcripts were identified as putative targets of the differentially expressed miRNAs. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis suggested that some of the differentially expressed miRNAs, including miR6435, miR5021 and miR1134, might be involved in terpenoid biosynthesis in the X. strumarium glandular trichomes. This study provides the first comprehensive analysis of miRNAs in X. strumarium, which forms the basis for further understanding of miRNA-based regulation on terpenoid biosynthesis.

GA3 and other signal regulators (MeJA and IAA) improve xanthumin biosynthesis in different manners in Xanthium strumarium L.

Xanthanolides from Xanthium strumarium L. exhibit various pharmacological activities and these compounds are mainly produced in the glandular trichomes of aerial plant parts. The regulation of xanthanolide biosynthesis has never been reported in the literature. In this study, the effects of phytohormonal stimulation on xanthumin (a xanthanolide compound) biosynthesis, glandular trichomes and germacrene A synthase (GAS) gene expression in X. strumarium L. young leaves were investigated. The exogenous applications of methyl jasmonate (MeJA), indole-3-acetic acid (IAA), and gibberrellin A3 (GA3) at appropriate concentrations were all found to improve xanthumin biosynthesis, but in different ways. It was suggested that a higher gland density stimulated by MeJA (400 µM) or IAA (200 µM) treatment caused at least in part an improvement in xanthumin production, whereas GA3 (10 µM) led to an improvement by up-regulating xanthumin biosynthetic genes within gland cells, not by forming more glandular trichomes. Compared to the plants before the flowering stage, plants that had initiated flowering showed enhanced xanthumin biosynthesis, but no higher gland density, an effect was similar to that caused by exogenous GA3 treatment.

How the novel integration of electrolysis in tidal flow constructed wetlands intensifies nutrient removal and odor control.

Intensified nutrient removal and odor control in a novel electrolysis-integrated tidal flow constructed wetland were evaluated. The average removal efficiencies of COD and NH4(+)-N were above 85% and 80% in the two experimental wetlands at influent COD concentration of 300 mg/L and ammonium nitrogen concentration of 60 mg/L regardless of electrolysis integration. Effluent nitrate concentration decreased from 2.5mg/L to 0.5mg/L with the reduction in current intensity from 1.5 mA/cm(2) to 0.57 mA/cm(2). This result reveals the important role of current intensity in nitrogen transformation. Owing to the ferrous and ferric iron coagulant formed through the electro-dissolution of the iron anode, electrolysis integration not only exerted a positive effect on phosphorus removal but also effectively inhibited sulfide accumulation for odor control. Although electrolysis operation enhanced nutrient removal and promoted the emission of CH4, no significant difference was observed in the microbial communities and abundance of the two experimental wetlands.

Intensified nitrogen and phosphorus removal in a novel electrolysis-integrated tidal flow constructed wetland system.

A novel electrolysis-integrated tidal flow constructed wetland (CW) system was developed in this study. The dynamics of intensified nitrogen and phosphorus removal and that of hydrogen sulphide control were evaluated. Ammonium removal of up to 80% was achieved with an inflow concentration of 60 mg/L in wetland systems with and without electrolysis integration. Effluent nitrate concentration decreased from 2 mg/L to less than 0.5 mg/L with the decrease in current intensity from 1.5 mA/cm(2) to 0.57 mA/cm(2) in the electrolysis-integrated wetland system, thus indicating that the current intensity of electrolysis plays an important role in nitrogen transformations. Phosphorus removal was significantly enhanced, exceeding 95% in the electrolysis-integrated CW system because of the in-situ formation of a ferric iron coagulant through the electro-dissolution of a sacrificial iron anode. Moreover, the electrolyzed wetland system effectively inhibits sulphide accumulation as a result of a sulphide precipitation coupled with ferrous-iron electro-dissolution and/or an inhibition of bacterial sulphate reduction under increased aerobic conditions.

Molecular cloning and functional characterization of two divergent 4-coumarate†:†coenzyme A ligases from Kudzu (Pueraria lobata).

As part of the efforts to understand isoflavonoid metabolism in Pueraria lobata at the molecular level, the cDNAs encoding two divergent 4-coumarate : coenzyme A ligases (4CLs, designated Pl4CL1 and Pl4CL2, respectively) were isolated from P. lobata roots. Sequence analysis revealed that Pl4CL1 had an N-terminal extension of twenty-one amino acid residues compared to Pl4CL2. Phylogenetic analysis showed that Pl4CL1 and Pl4CL2 fell into angiosperm Class II and Class I, respectively. Through in vitro biochemical assays, both Pl4CLs were found to have the capacity to utilize 4-coumarate and trans-cinnamate as substrates, while neither of them could convert sinapate. Pl4CL2 had a broader substrate specificity than Pl4CL1. The affinity of Pl4CL1 for 4-coumarate was 2.6-fold higher than that of Pl4CL2 (with the Km values of 3.5 µM and 9.1 µM, respectively). Combining the dataset including gene expression profiles, metabolites measurements, and biochemical properties, our results indicated that Pl4CL1, just as other angiosperm Class II 4CLs, might play a role in isoflavone biosynthesis in P. lobata, while Pl4CL2 belongs to angiosperm Class I, and may function as a housekeeping enzyme concerning lignification.

Identifying three ecological chemotypes of Xanthium strumarium glandular trichomes using a combined NMR and LC-MS method.

Xanthanolides, as the sesquiterpene lactones, are reportedly the major components for the pharmacological properties of X. strumarium L. species. Phytochemical studies indicated that the glandular structures on the surface of plant tissues would form the primary sites for the accumulation of this class of the compounds. As the interface between plants and their natural enemies, glandular trichomes may vary with respect to which of their chemicals are sequestered against different herbivores in different ecologies. However, to date, no data are available on the chemical characterisation of X. strumarium glandular cells. In this study, the trichome secretions of the X. strumarium species originating from nineteen unique areas across eleven provinces in China, were analysed by HPLC, LC-ESI-MS and NMR. For the first time three distinct chemotypes of X. strumarium glandular trichomes were discovered along with the qualitative and quantitative evaluations of their presence of xanthanolides; these were designated glandular cell Types I, II, and III, respectively. The main xanthanolides in Type I cells were 8-epi-xanthatin and xanthumin while no xanthatin was detected. Xanthatin, 8-epi-xanthatin, and xanthumin dominated in Type II cells with comparable levels of each being present. For Type III cells, significantly higher concentrations of 8-epi-xanthatin or xanthinosin (relative to xanthatin) were detected with xanthinosin only being observed in this type. Further research will focus on understanding the ecological and molecular mechanism causing these chemotype differences in X. strumarium glandular structures.