Transcriptome analysis provides insights into coumarin biosynthesis in the medicinal plant Angelica dahurica cv. Yubaizhi.

Angelica dahurica roots have a long history of use in traditional Chinese medicine due to their high coumarin content. To address the increasing demand for these roots, a synthetic biology approach has been proposed. Nevertheless, our comprehension of coumarin biosynthesis and its regulation remains limited. In this study, we utilized Hiseq2500 sequencing to analyze the transcriptomes of A. dahurica at different growth stages while concurrently quantifying coumarin content. Differentially expressed gene (DEG) analysis was employed to identify key genes involved in coumarin and terpenoid backbone biosynthesis. Weighted gene co-expression network analysis (WGCNA) was applied to identify gene modules strongly associated with coumarin content, elucidating the regulatory relationships between transcription factors (TFs) and pathway genes. Furthermore, KEGG enrichment analysis was used to explore essential pathways governing coumarin biosynthesis, with the identification of hub genes. Our results indicated that total coumarin content was highest in the roots, followed by leaves and stems, across all three developmental stages. Transcriptome analysis identified a total of 92,478 genes, among which 215 and 30 genes were implicated in coumarin and terpenoid backbone biosynthesis, respectively. Within the 73 identified gene modules by WGCNA, three modules-namely aquamarine1 (comprising two OMTs, one CSE, one AACT, one HDS, two PSs, one 2OGO, four UGTs, and seven CYP450s), darkmagenta (containing one UGT and one HDR), and navajowhite2 (consisting of one HCT, three UGTs, one CYP71A25, one OMT, one CSE, one HDS, and one PT)-were strongly associated with imperatorin, oxypeucedanin, and isoimperatorin content, respectively. KEGG enrichment analysis highlighted significant enrichment of cytochrome P450, transporter, and ubiquitin system pathways. Moreover, TF-gene regulatory analysis unveiled the complexity of coumarin biosynthesis, with 17 TF families regulating 17 genes in the aquamarine1 module, 8 TF families regulating 2 genes in the darkmagenta module, and 8 TF families regulating 7 genes in the navajowhite2 module. These comprehensive findings provide valuable insights into coumarin biosynthesis in A. dahurica, facilitating future research and potential applications in traditional Chinese medicine and synthetic biology strategies.

Metabolic characteristics of self-pollinated wheat seed under red and blue light during early development.

MAIN CONCLUSION: Blue light has a greater effect on jasmonic acid and flavonoid accumulation in wheat seeds than red light; blue light reduces starch synthesis and the size of starch granules and seeds. This study sought to elucidate the effects of blue and red light on seed metabolism to provide important insights regarding the role of light quality in regulating seed growth and development. We used combined multi-omics analysis to investigate the impact of red and blue light (BL) on the induction of secondary metabolite accumulation in the hexaploid wheat Dianmai 3 after pollination. Flavonoids and alkaloids were the most differentially abundant metabolites detected under different treatments. Additionally, we used multi-omics and weighted correlation network analysis to screen multiple candidate genes associated with jasmonic acid (JA) and flavonoids. Expression regulatory networks were constructed based on RNA-sequencing data and their potential binding sites. The results revealed that BL had a greater effect on JA and flavonoid accumulation in wheat seeds than red light. Furthermore, BL reduced starch synthesis and stunted the size of starch granules and seeds. Collectively, these findings clarify the role of BL in the metabolic regulation of early seed development in wheat.

Immunogenicity and protective effects of recombinant bivalent COVID-19 vaccine in mice and rhesus macaques.

Coronavirus disease (COVID-19) is still spreading rapidly worldwide, and a safe, effective, and cheap vaccine is still required to combat the COVID-19 pandemic. Here, we report a recombinant bivalent COVID-19 vaccine containing the RBD proteins of the prototype strain and beta variant. Immunization studies in mice demonstrated that this bivalent vaccine had far greater immunogenicity than the ZF2001, a marketed monovalent recombinant protein COVID-19 vaccine, and exhibited good immunization effects against the original COVID-19 strain and various variants. Rhesus macaque challenge experiments showed that this bivalent vaccine drastically decreased the lung viral load and reduced lung lesions in SARS-CoV-2 (the causative virus of COVID-19)-infected rhesus macaques. In summary, this bivalent vaccine showed immunogenicity and protective efficacy that was far superior to the monovalent recombinant protein vaccine against the prototype strain and provided an important basis for developing broad-spectrum COVID-19 vaccines.

Development of HPV58 type-specific antibodies and detection kit.

Currently, human papillomavirus (HPV) vaccines are in short supply, so the development of HPV vaccines has a broad market prospect. The 3-, 9-, and 15-valent HPV vaccines developed by ourselves all contain HPV58-derived antigen components. It is important to detect HPV 58 during vaccine production. Here, we introduced a development process of HPV58 type-specific antibodies and a detection kit. Briefly, HPV58 L1-Virus Like Particles (VLPs) were used as antigens to immunize mice, followed by extraction of the ascites to prepare hybridoma cells. After culturing, the supernatants containing secreted antibodies were harvested, purified, and screened to obtain monoclonal antibodies (mAbs). In the pool of attained monoclonal antibodies, we selected 2F7 and 2G7 to evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity and gene sequencing. Finally, an enzyme-linked immunosorbent assay (ELISA) detection kit was assembled with 2F7 and 2G7 mAbs which possessed high specificity to HPV58 L1-VLPs. The detection kit developed by 2F7 and 2G7 could be adopted to specifically detect HPV58 L1 protein with good linearity and detection range, which could be widely used in clinical testing and quality control in the production of HPV vaccines.Abbreviations: BSA: Bovine serum albumin; CDRs: Complementarity-determining regions; CV: Coefficient of variation; DTT: Dithiothreitol; ELISA: Enzyme-linked immunosorbent assay; HAT: Hypoxanthine-aminopterin-thymidine; HPV: Human Papillomavirus; IC50: 50% inhibition rate; IC90: 90% inhibition rate; mAbs: Monoclonal antibodies; VLP: Virus-like particle.

Development and characterization of anti-HPV16 monoclonal antibodies for assembly of an HPV16 detection kit.

Quality control is very important during the development of 3-valent (16/18/58), 9-valent (6/11/16/18/31/33/45/52/58), and 15-valent human papillomavirus (HPV) vaccines (6/11/16/18/31/33/35/39/45/52/56/58/59/68). All 3-valent, 9-valent, and 15-valent HPV vaccines contain the HPV16 antigen; therefore, a detection method that can specifically identify HPV16 in vaccines is urgently required. This study aimed to develop and characterize monoclonal antibodies to assemble a highly specific HPV16 detection kit. The HPV16 L1 pentameric protein developed as an immunogen was used to prepare monoclonal antibodies. From the pool of prepared monoclonal antibodies, we selected 4G12 and 5A6 to screen and evaluate their subtypes, specificity, neutralizing activity, serum competition, binding affinity, and gene sequencing. After these characterizations, an enzyme-linked immunosorbent assay kit for these monoclonal antibodies was developed, and excellent quality was demonstrated in the assessment of linearity, repeatability, and specificity. The developed detection kit has great potential for wide use in clinical testing and quality control in vaccine production processes.

Transcriptome and Metabolome Combined to Analyze Quinoa Grain Quality Differences of Different Colors Cultivars.

Quinoa (Chenopodium quinoa Wild.) has attracted considerable attention owing to its unique nutritional, economic, and medicinal values. Meanwhile, quinoa germplasm resources and grain colors are rich and diverse. In this study, we analyzed the composition of primary and secondary metabolites and the content of the grains of four different high-yield quinoa cultivars (black, red, white, and yellow) harvested 42 days after flowering. The grains were subjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and transcriptome sequencing to identify the differentially expressed genes and metabolites. Analysis of candidate genes regulating the metabolic differences among cultivars found that the metabolite profiles differed between white and black quinoa, and that there were also clear differences between red and yellow quinoa. It also revealed significantly altered amino acid, alkaloid, tannin, phenolic acid, and lipid profiles among the four quinoa cultivars. Six common enrichment pathways, including phenylpropane biosynthesis, amino acid biosynthesis, and ABC transporter, were common to metabolites and genes. Moreover, we identified key genes highly correlated with specific metabolites and clarified the relationship between them. Our results provide theoretical and practical references for breeding novel quinoa cultivars with superior quality, yield, and stress tolerance. Furthermore, these findings introduce an original approach of integrating genomics and transcriptomics for screening target genes that regulate the desirable traits of quinoa grain.

Integrating transcriptomics and metabolomics to analyze quinoa (Chenopodium quinoa Willd.) responses to drought stress and rewatering.

The crop production of quinoa (Chenopodium quinoa Willd.), the only plant meeting basic human nutritional requirements, is affected by drought stress. To better understand the drought tolerance mechanism of quinoa, we screened the drought-tolerant quinoa genotype "Dianli 129" and studied the seedling leaves of the drought-tolerant quinoa genotype after drought and rewatering treatments using transcriptomics and targeted metabolomics. Drought-treatment, drought control, rewatering-treated, and rewatered control were named as DR, DC, RW, and RC, respectively. Among four comparison groups, DC vs. DR, RC vs. RW, RW vs. DR, and RC vs. DC, we identified 10,292, 2,307, 12,368, and 3 differentially expressed genes (DEGs), and 215, 192, 132, and 19 differentially expressed metabolites (DEMs), respectively. A total of 38,670 genes and 142 pathways were annotated. The results of transcriptome and metabolome association analysis showed that gene-LOC110713661 and gene-LOC110738152 may be the key genes for drought tolerance in quinoa. Some metabolites accumulated in quinoa leaves in response to drought stress, and the plants recovered after rewatering. DEGs and DEMs participate in starch and sucrose metabolism and flavonoid biosynthesis, which are vital for improving drought tolerance in quinoa. Drought tolerance of quinoa was correlated with gene expression differences, metabolite accumulation and good recovery after rewatering. These findings improve our understanding of drought and rewatering responses in quinoa and have implications for the breeding of new drought-tolerance varieties while providing a theoretical basis for drought-tolerance varieties identification.

Exploring and applying genes to enhance the resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) is a destructive disease in wheat worldwide. Fusarium graminearum species complex (FGSC) is the main causal pathogen causing severe damage to wheat with reduction in both grain yield and quality. Additionally, mycotoxins produced by the FHB pathogens are hazardous to the health of human and livestock. Large numbers of genes conferring FHB resistance to date have been characterized from wheat and its relatives, and some of them have been widely used in breeding and significantly improved the resistance to FHB in wheat. However, the disease spreads rapidly and has been severe due to the climate and cropping system changes in the last decade. It is an urgent necessity to explore and apply more genes related to FHB resistant for wheat breeding. In this review, we summarized the genes with FHB resistance and mycotoxin detoxication identified from common wheat and its relatives by using forward- and reverse-genetic approaches, and introduced the effects of such genes and the genes with FHB resistant from other plant species, and host-induced gene silencing (HIGS) in enhancing the resistance to FHB in wheat. We also outlined the molecular rationale of the resistance and the application of the cloned genes for FHB control. Finally, we discussed the future challenges and opportunities in this field.

Combined transcriptome and metabolome analysis of the resistance mechanism of quinoa seedlings to Spodoptera exigua.

Quinoa has attracted considerable attention owing to its unique nutritional, economic, and medicinal values. The damage intensity of Spodoptera exigua at the seedling stage of quinoa fluctuates with the crop's biological cycle and the environmental changes throughout the growing season. In this study, we used independently selected quinoa seedling resistant and susceptible cultivars to investigate the difference between insect resistance and insect susceptibility of quinoa at the seedling stage. Samples were collected when Spodoptera exigua 45 days after planting the seedlings, and broad targeted metabolomics studies were conducted using liquid chromatography-mass spectrophotometry combined with transcriptomic co-analysis. The metabolomic and genomic analyses of the insect-resistant and insect-susceptible quinoa groups revealed a total of 159 differential metabolites and were functionally annotated to 2334 differential genes involved in 128 pathways using the Kyoto Encyclopedia of Genes and Genomes analysis. In total, 14 metabolites and 22 genes were identified as key factors for the differential accumulation of insect-resistant metabolites in quinoa seedlings. Among them, gene-LOC110694254, gene-LOC110682669, and gene-LOC110732988 were positively correlated with choline. The expression of gene-LOC110729518 and gene-LOC110723164, which were notably higher in the resistant cultivars than in the susceptible cultivars, and the accumulations of the corresponding metabolites were also significantly higher in insect-resistant cultivars. These results elucidate the regulatory mechanism between insect resistance genes and metabolite accumulation in quinoa seedlings, and can provide a basis for the breeding and identification of new insect-resistant quinoa cultivars as well as for screening potential regulatory metabolites of quinoa insect-resistant target genes.

Transcriptomics and metabolomics analyses of the mechanism of flavonoid synthesis in seeds of differently colored quinoa strains.

Quinoa (Chenopodium quinoa Willd.) is an herb of the genus Chenopodiaceae that is native to the Andes Mountains of South America. To understand the metabolic differences between various quinoa strains, we selected quinoa strains of four colors (black, red, yellow, and white) and we subjected seeds to extensive targeted metabolomics analysis using liquid chromatography-tandem mass spectrometry and transcriptomics analysis. In total, 90 flavonoid-related metabolites were detected in quinoa seeds of the four colors. We elucida ted the regulatory mechanisms of flavonoid biosynthesis in the different quinoa varieties, and thus identified key genes for flavonoid biosynthesis. The results showed that 18 flavone metabolites and 25 flavonoid-related genes were key contributors to flavonoid biosynthesis in quinoa seeds. The results of this study may provide a basis for the breeding and identification of new quinoa strains and for the screening of potential target genes in flavonoid biosynthesis regulation in quinoa.

Transcriptomics Integrated With Widely Targeted Metabolomics Reveals the Mechanism Underlying Grain Color Formation in Wheat at the Grain-Filling Stage.

Colored wheat grains have a unique nutritional value. To elucidate the color formation mechanism in wheat seeds, comprehensive metabolomic and transcriptomic analyses were conducted on purple (Dianmai 20-1), blue (Dianmai 20-8), and white (Dianmai 16) wheat at the grain-filling stage. The results showed that the flavonoid biosynthesis pathway was closely related to grain color formation. Among the 603 metabolites identified in all varieties, there were 98 flavonoids. Forty-six flavonoids were detected in purple and blue wheat, and there were fewer flavonoids in white wheat than in colored wheat. Integrated transcriptomic and metabolomic analyses showed that gene expression modulated the flavonoid composition and content, resulting in different metabolite levels of pelargonidin, cyanidin, and delphinidin, thus affecting the color formation of wheat grains. The present study clarifies the mechanism by which pigmentation develops in wheat grains and provides an empirical reference for colored wheat breeding.

Cross-neutralizing antibody titres against non-vaccine types induced by a recombinant trivalent HPV vaccine (16/18/58) in rhesus macaques.

Human papillomavirus (HPV) causes not only most cervical cancers but also cancers of the vagina, vulva, penis, anus, rectum, and oropharynx. Every year, 200,000 women die of cervical cancer in the world, and China accounts for about 10%. HPV vaccines are effective in preventing HPV infections thus HPV-related cancers worldwide. Studies on the clinical trials of the 2v Cervarix™ and the 4v Gardasil® have suggested that immunization with either of these vaccines provided some level of protection against other HPV types that are closely related to the types contained in the vaccines. Here we conducted a preliminary evaluation on the ability to induce cross-neutralizing antibodies in rhesus monkeys by a 3v HPV vaccine that targets HPV16, 18, and 58 and it is specifically designed for Chinese women. We found that this vaccine is no less than Gardasil® in terms of the ability to induce NAbs against non-vaccine types of HPV in rhesus macaques. These results provided evidence from the immunogenicity point of view that the KLWS 3v HPV vaccine is a strong competitor to the imported 2v and 4v HPV vaccines currently available on the market.

Non-targeted metabolomics of quinoa seed filling period based on liquid chromatography-mass spectrometry.

Quinoa (Chenopodium quinoa Willd.), an herb belonging to the amaranth family, is rich in minerals, amino acids, vitamins, proteins, and flavonoids. Its grain, compared with other major grains, has unique nutritional value with tremendous applications. This study used four independently bred high-generation lines (seed colors) of quinoa as materials to further understand the metabolic differences in the filling periods of quinoa varieties. Additionally, the non-targeted metabolome of quinoa seeds 35 and 42 days after flowering, respectively, were studied via liquid chromatography-mass spectrometry. The two filling periods of yellow, white, black, and red quinoa grains resulted in significant differences in the metabolites, particularly in L-methionine, S-adenosyl-L-homocysteine, S-adenosyl-L-methionine, pyruvate, fumarate, and oxaloacetate. Soluble sugar, amino acid, and fatty acid contents in quinoa increased after 42 days of flowering. There were metabolic differences between the sugar phosphates (L-fucose, D-mannose-6-phosphate, xylulose-5-phosphate, sedoheptulose-7-phosphate), amino acid (alanine), and organic compounds (kynurenate, tryptamine, serotonin, bilirubin) among the four quinoa varieties. The relative difference in the metabolites was largest when the yellow quinoa grain was compared with the other quinoa varieties and smallest when the red and black varieties were compare. The results of this study provide a basis for the reproduction and identification of new quinoa varieties, as well as for screening potential quality control target genes by combining genomics and transcriptomics.

Total synthesis of (±)-epi-stemodan-13α,17-diol.

Stemodan-13α,17-diol is a natural stemodane-type diterpenoid isolated from Stemodia chilensis. Herein we report the total synthesis of its epimer, stemodan-13ß,17-diol, by applying titanium-mediated polyene cyclization and iron-catalyzed [5 + 2] cycloaddition as the key transformations to expeditiously install the molecular scaffold.

A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses.

Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 µm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 µm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.

Development and characterization of an HPV18 detection kit using two novel HPV18 type-specific monoclonal antibodies.

BACKGROUND: HPV 18 is one of the most prevalent oncogenic types, only second to HPV 16, and included in the licensed vaccines on the market. In this study, we describe the production and characterization of a panel of monoclonal antibodies (mAb) to HPV18. METHODS: The immunocompetence of 1B1 and 4C2 mAbs for HPV L1 protein was evaluated by SDS-PAGE analysis, neutralization assays, affinity identification, and ELISA. The 1B1 and 4C2 genes were sequenced and analyzed. Finally, the detection kit with the two mAbs was assessed for linearity, repeatability and specificity. RESULTS: Both mAbs specifically recognized HPV18 L1 and virus-like particles (VLPs). These mAbs are conformation-neutralizing antibodies that have high affinity and type specificity. Based on these characteristics of these mAbs, we developed an ELISA kit for specifically detecting HPV 18 antigen. We showed that this kit displayed good linearity, repeatability and sensitivity for detecting HPV18 L1 pentamer and HPV18 VLP. CONCLUSIONS: We characterized two monoclonal neutralizing antibodies for HPV L1 protein, and developed an ELISA kit for specifically detecting HPV 18 antigen. This newly developed kit can be used to monitor the potency of HPV vaccines throughout the entire production process as well as preliminary analysis of HPV18 infections.

Iron-Catalyzed Intramolecular Perezone-Type [5 + 2] Cycloaddition: Access to Tricyclo[6.3.1.01,6]dodecane.

An iron-catalyzed perezone-type [5 + 2] cycloaddition toward tricyclo[6.3.1.01,6]dodecane scaffolds is presented, furnishing cycloadducts with two new C-C bonds and three to four stereogenic centers generated with typically good yields and excellent diastereoselectivities. Two independent conditions, catalytic FeCl3/PhCO3 tBu (0.5 equiv/1 equiv) and stoichiometric FeCl3(2 equiv), have been respectively manifested to be applicable for various substrates. Mechanistically, the reaction may proceed via iron-mediated generation of a carbon cationic center, which triggers subsequent [5 + 2] cyclization. Derivation of the cycloadduct was conducted to testify the applicability of this method.

A novel trivalent HPV 16/18/58 vaccine with anti-HPV 16 and 18 neutralizing antibody responses comparable to those induced by the Gardasil quadrivalent vaccine in rhesus macaque model.

Persistent infection with human papillomavirus (HPV) is a key factor in the development of precancerous lesions and invasive cervical cancer. Prophylactic vaccines to immunize against HPV are an effective approach to reducing HPV related disease burden. In this study, we investigated the immunogenicity and dosage effect of a trivalent HPV 16/18/58 vaccine (3vHPV) produced in Escherichia coli (E.coli), with Gardasil quadrivalent vaccine (4vHPV, Merck & Co.) as a positive control. Sera collected from rhesus macaques vaccinated with three dosage formulations of 3vHPV (termed low-, mid-, and high-dosage formulations, respectively), and the 4vHPV vaccine were analyzed by both Pseudovirus-Based Neutralization Assay (PBNA) and Enzyme-Linked Immunosorbent Assay (ELISA). Strong immune responses against HPV 16/18/58 were successfully elicited, and dosage-dependence was observed, with likely occurrence of immune interference between different L1-VLP antigens. HPV 16/18 specific neutralizing antibody (nAb) and total immunoglobulin G (IgG) antibody responses in rhesus macaques receiving 3vHPV at the three dosages tested were generally non-inferior to those observed in rhesus macaques receiving 4vHPV throughout the study period. Particularly, HPV 18 nAb titers induced by the mid-dosage formulation that contained the same amounts of HPV 16/18 L1-VLPs as Gardasil 4vHPV were between 7.3 to 12.7-fold higher compared to the positive control arm from weeks 24-64. The durability of antibody responses specific to HPV 16/18 elicited by 3vHPV vaccines was also shown to be non-inferior to that associated with Gardasil 4vHPV.