Karyotyping of aneuploid and polyploid plants from low coverage whole-genome resequencing.

BACKGROUND: Karyotype, as a basic characteristic of species, provides valuable information for fundamental theoretical research and germplasm resource innovation. However, traditional karyotyping techniques, including fluorescence in situ hybridization (FISH), are challenging and low in efficiency, especially when karyotyping aneuploid and polyploid plants. The use of low coverage whole-genome resequencing (lcWGR) data for karyotyping was explored, but existing methods are complicated and require control samples. RESULTS: In this study, a new protocol for molecular karyotype analysis was provided, which proved to be a simpler, faster, and more accurate method, requiring no control. Notably, our method not only provided the copy number of each chromosome of an individual but also an accurate evaluation of the genomic contribution from its parents. Moreover, we verified the method through FISH and published resequencing data. CONCLUSIONS: This method is of great significance for species evolution analysis, chromosome engineering, crop improvement, and breeding.

Downregulation of the expression of subgenomic chromosome A7 genes promotes plant height in resynthesized allopolyploid Brassica napus.

KEY MESSAGE: Homoeolog expression bias and the gene dosage effect induce downregulation of genes on chromosome A7, causing a significant increase in the plant height of resynthesized allopolyploid Brassica napus. Gene expression levels in allopolyploid plants are not equivalent to the simple average of the expression levels in the parents and are associated with several non-additive expression phenomena, including homoeolog expression bias. However, hardly any information is available on the effect of homoeolog expression bias on traits. Here, we studied the effects of gene expression-related characteristics on agronomic traits using six isogenic resynthesized Brassica napus lines across the first ten generations. We found a group of genes located on chromosome A7 whose expression levels were significantly negatively correlated with plant height. They were expressed at significantly lower levels than their homoeologous genes, owing to allopolyploidy rather than inheritance from parents. Homoeolog expression bias resulted in resynthesized allopolyploids with a plant height similar to their female Brassica oleracea parent, but significantly higher than that of the male Brassica rapa parent. Notably, aneuploid lines carrying monosomic and trisomic chromosome A7 had the highest and lowest plant heights, respectively, due to changes in the expression bias of homoeologous genes because of alterations in the gene dosage. These findings suggest that the downregulation of the expression of homoeologous genes on a single chromosome can result in the partial improvement of traits to a significant extent in the nascent allopolyploid B. napus.

Characterizations of microRNAs involved in the molecular mechanisms underlying the therapeutic effects of noni (Morinda citrifolia L.) fruit juice on hyperuricemia in mice.

Background: Hyperuricemia is generally defined as the high level of serum uric acid and is well known as an important risk factor for the development of various medical disorders. However, the medicinal treatment of hyperuricemia is frequently associated with multiple side-effects. Methods: The therapeutic effect of noni (Morinda citrifolia L.) fruit juice on hyperuricemia and the underlying molecular mechanisms were investigated in mouse model of hyperuricemia induced by potassium oxonate using biochemical and high-throughput RNA sequencing analyses. Results: The levels of serum uric acid (UA) and xanthine oxidase (XOD) in mice treated with noni fruit juice were significantly decreased, suggesting that the noni fruit juice could alleviate hyperuricemia by inhibiting the XOD activity and reducing the level of serum UA. The contents of both serum creatinine and blood urine nitrogen of the noni fruit juice group were significantly lower than those of the model group, suggesting that noni fruit juice promoted the excretion of UA without causing deleterious effect on the renal functions in mice. The differentially expressed microRNAs involved in the pathogenesis of hyperuricemia in mice were identified by RNA sequencing with their target genes further annotated based on both Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases to explore the metabolic pathways and molecular mechanisms underlying the therapeutic effect on hyperuricemia by noni fruit juice. Conclusion: Our study provided strong experimental evidence to support the further investigations of the potential application of noni fruit juice in the treatment of hyperuricemia.

Molecular and biochemical investigations of the anti-fatigue effects of tea polyphenols and fruit extracts of Lycium ruthenicum Murr. on mice with exercise-induced fatigue.

Background: The molecular mechanisms regulating the therapeutic effects of plant-based ingredients on the exercise-induced fatigue (EIF) remain unclear. The therapeutic effects of both tea polyphenols (TP) and fruit extracts of Lycium ruthenicum (LR) on mouse model of EIF were investigated. Methods: The variations in the fatigue-related biochemical factors, i.e., lactate dehydrogenase (LDH), superoxide dismutase (SOD), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-2 (IL-2), and interleukin-6 (IL-6), in mouse models of EIF treated with TP and LR were determined. The microRNAs involved in the therapeutic effects of TP and LR on the treatment of mice with EIF were identified using the next-generation sequencing technology. Results: Our results revealed that both TP and LR showed evident anti-inflammatory effect and reduced oxidative stress. In comparison with the control groups, the contents of LDH, TNF-α, IL-6, IL-1ß, and IL-2 were significantly decreased and the contents of SOD were significantly increased in the experimental groups treated with either TP or LR. A total of 23 microRNAs (21 upregulated and 2 downregulated) identified for the first time by the high-throughput RNA sequencing were involved in the molecular response to EIF in mice treated with TP and LR. The regulatory functions of these microRNAs in the pathogenesis of EIF in mice were further explored based on Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses with a total of over 20,000-30,000 target genes annotated and 44 metabolic pathways enriched in the experimental groups based on GO and KEGG databases, respectively. Conclusion: Our study revealed the therapeutic effects of TP and LR and identified the microRNAs involved in the molecular mechanisms regulating the EIF in mice, providing strong experimental evidence to support further agricultural development of LR as well as the investigations and applications of TP and LR in the treatment of EIF in humans, including the professional athletes.

Identification of potato Lipid transfer protein gene family and expression verification of drought genes StLTP1 and StLTP7.

Lipid transfer proteins (LTPs) are widely distributed in plants and play an important role in the response to stress. Potato (Solanum tuberosum L.) is sensitive to a lack of water, and drought stress is one of the limiting factors for its yield. Therefore, mining candidate functional genes for drought stress and creating new types of potato germplasm for drought resistance is an effective way to solve this problem. There are few reports on the LTP family in potato. In this study, 39 members of the potato LTP family were identified. They were located on seven chromosomes, and the amino acid sequences encoded ranged from 101 to 345 aa. All 39 family members contained introns and had exons that ranged from one to four. Conserved motif analysis of potato LTP transcription factors showed that 34 transcription factors contained Motif 2 and Motif 4, suggesting that they were conserved motifs of potato LTP. Compared with the LTP genes of homologous crops, the potato and tomato (Solanum lycopersicum L.) LTPs were the mostly closely related. The StLTP1 and StLTP7 genes were screened by quantitative reverse transcription PCR combined with potato transcriptome data to study their expression in tissues and the characteristics of their responses to drought stress. The results showed that StLTP1 and StLTP7 were upregulated in the roots, stems, and leaves after PEG 6000 stress. Taken together, our study provides comprehensive information on the potato LTP family that will help to develop a framework for further functional studies.

Self-assembling ferritin nanoparticles coupled with linear sequences from canine distemper virus haemagglutinin protein elicit robust immune responses.

BACKGROUND: Canine distemper virus (CDV), which is highly infectious, has caused outbreaks of varying scales in domestic and wild animals worldwide, so the development of a high-efficiency vaccine has broad application prospects. Currently, the commercial vaccine of CDV is an attenuated vaccine, which has the disadvantages of a complex preparation process, high cost and safety risk. It is necessary to develop a safe and effective CDV vaccine that is easy to produce on a large scale. In this study, sequences of CDV haemagglutinin (HA) from the Yanaka strain were aligned, and three potential linear sequences, termed YaH3, YaH4, and YaH5, were collected. To increase the immunogenicity of the epitopes, ferritin was employed as a self-assembling nanoparticle element. The ferritin-coupled forms were termed YaH3F, YaH4F, and YaH5F, respectively. A full-length HA sequence coupled with ferritin was also constructed as a DNA vaccine to compare the immunogenicity of nanoparticles in prokaryotic expression. RESULT: The self-assembly morphology of the proteins from prokaryotic expression was verified by transmission electron microscopy. All the proteins self-assembled into nanoparticles. The expression of the DNA vaccine YaHF in HEK-293T cells was also confirmed in vitro. After subcutaneous injection of epitope nanoparticles or intramuscular injection of DNA YaHF, all vaccines induced strong serum titres, and long-term potency of antibodies in serum could be detected after 84 days. Strong anti-CDV neutralizing activities were observed in both the YaH4F group and YaHF group. According to antibody typing and cytokine detection, YaH4F can induce both Th1 and Th2 immune responses. The results of flow cytometry detection indicated that compared with the control group, all the immunogens elicited an increase in CD3. Simultaneously, the serum antibodies induced by YaH4F and YaHF could significantly enhance the ADCC effect compared with the control group, indicating that the antibodies in the serum effectively recognized the antigens on the cell surface and induced NK cells to kill infected cells directly. CONCLUSIONS: YaH4F self-assembling nanoparticle obtained by prokaryotic expression has no less of an immune effect than YaHF, and H4 has great potential to become a key target for the easy and rapid preparation of epitope vaccines.

Comparative analysis of the main medicinal substances and applications of Echium vulgare L. and Echium plantagineum L.: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Echium vulgare L. and Echium plantagineum L. originated in the Mediterranean, and were later domesticated in Africa, America, Asia, Europe and Oceania, where they were widely used to treat many diseases including cough, urinary tract infection, fever, inflammation and muscle strain. AIM OF THE STUDY: The purpose of this review is to provide scientific literature on the traditional uses, bioactive chemical components and pharmacological activities of two species of Echium, and to critically analyze the information provided, so as to understand the current work on these two species and explore the possible prospect of this plant in pharmaceutical research. METHODS: Systematic review and meta-analysis were conducted according to Prisma guidelines, and the related literatures searched on Google Academic, Science Direct, Baidu Scholars and China National Knowledge Infrastructure (CNKI) up to June 2021 were reviewed. The key words used are: Echium, E.vulgare, E.plantagineum, plant components, chemical components, pharmacological activities, pharmaceutical products and applications. Thereafter all eligible studies are analyzed and summarized in this review. The selection of manuscripts is based on the following inclusion criteria: the article has years of research or publication, is published in English, Portuguese or Spanish and Chinese, and there are keywords in the title, abstract, keywords or full text of the article. For the selection of manuscripts, first, select articles according to titles, then summarize them, and finally, analyze the full text of the publication. Elimination criteria: 1. Duplicate reports; 2. There are research design defects and poor quality; 3. Incomplete data and unclear ending effect; 4. The statistical method is wrong and cannot be corrected. RESULTS: The pharmacological characteristics of E.vulgare and E.plantagineum can basically support their traditional use, but the medicinal substances contained in them are quite different in composition and content, and the development and application of corresponding products are also different. CONCLUSIONS: At present, there is little clinical data about drugs related to the two species, and more research is needed in the future, especially human experiments and clinical trials, to evaluate the cellular and molecular mechanisms based on pharmacological, biological activity and safety studies, and to provide more powerful scientific basis for their traditional medicinal properties. In addition, the further application and development of the medicinal products of E.vulgare and E.plantagineum still need to be precise and identified, so as to give full play to their medicinal potential.

MicroRNAs Involved in the Therapeutic Functions of Noni (Morinda citrifolia L.) Fruit Juice in the Treatment of Acute Gouty Arthritis in Mice Induced with Monosodium Urate.

We investigated the functions of microRNAs in the therapeutic effects of noni (Morinda citrifolia L.) fruit juice on mouse models of acute gouty arthritis induced with monosodium urate (MSU). Compared with the model group (treated with MSU), mice in both the positive control group (treated with both MSU and colchicine) and noni fruit juice group (treated with MSU and noni fruit juice) showed a significantly decreased degree of paw swelling in 5 days, as well as the contents of two types of proinflammatory cytokines (i.e., NALP3 and TNF-α). Based on the next-generation sequencing technology, a total of 3896 microRNAs (234 known and 3662 novel) were identified in mice treated with noni fruit juice. A large amount of differentially expressed miRNAs were identified in the noni fruit juice group, suggesting the significant effects of noni fruit juice on the mice with acute gouty arthritis, while the different patterns of change in the numbers of both upregulated and downregulated miRNAs in both noni fruit juice and positive control groups indicated that the mice of acute gouty arthritis may be regulated by differential mechanisms between the treatments of noni fruit juice and colchicine. The target genes of microRNAs involved in the pathogenesis and pathology of acute gouty arthritis in mice were identified and further annotated by both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Our results revealed the therapeutic effects of noni fruit juice on acute gouty arthritis in mice with a group of microRNAs involved in the pharmacological mechanisms of noni fruit juice, providing scientific evidence to support both the agricultural cultivation and pharmacological significance of noni plants.

Self-assembled raccoon dog parvovirus VP2 protein confers immunity against RDPV disease in raccoon dogs: in vitro and in vivo studies.

BACKGROUND: Raccoon dog parvovirus (RDPV) causes acute infectious diseases in raccoon dogs and may cause death in severe cases. The current treatment strategy relies on the extensive usage of classical inactivated vaccine which is marred by large doses, short immunization cycles and safety concerns. METHODS: The present study aimed at optimization of RDPV VP2 gene, subcloning the gene into plasmid pET30a, and its subsequent transfer to Escherichia coli with trigger factor 16 for co-expression. The protein thus expressed was purified with ammonium sulfate precipitation, hydrophobic chromatography, and endotoxin extraction procedures. VLPs were examined by transmission electron microscopy, dynamic light scattering, and the efficacy of VLPs vaccine was tested in vivo. RESULTS: Results indicated that RDPV VP2 protein could be expressed soluble. Transmission electron microscopy and dynamic light scattering results indicated that RDPV VP2 self-assembled into VLPs. Hemagglutination inhibition antibody titers elicited by Al(OH)3 adjuvanted RDPV VLPs were comparable with RDPV inactivated vaccines, and the viral loads in the blood of the struck raccoon dogs were greatly reduced. Hematoxylin and eosin and Immunohistochemical results indicated that RDPV VLPs vaccine could protect raccoon dogs against RDPV infections. CONCLUSIONS: These results suggest that RDPV VLPs can become a potential vaccine candidate for RDPV therapy.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8.

BACKGROUND: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. OBJECTIVES: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. METHODS: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. RESULTS: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. CONCLUSIONS: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses.

Plant carotenoid cleavage oxygenase (CCO) is an enzyme that catalyzes the synthesis of carotenoids and participates in many important physiological functions. The plant CCOs exist in two forms, namely carotenoid cleavage dioxygenase (CCD) and nine-cis epoxide carotenoid dioxygenase (NCED). Although studies have shown that this gene family has been identified in many species, such as Arabidopsis, grape, and tomato, the evolutionary origin of the CCO family and the expression pattern of pepper genes in response to H2O2 and other abiotic stresses are still unclear. In this study, we used the bioinformatics method to identify and analyze the members of the CCO gene family from pepper and other 13 plants from lower to higher plant species based on the whole genome sequence. A total of 158 CCO genes were identified in different plant species and further divided into two groups (e.g., groups I and II). The former was subdivided into CCD7 and CCD8 and have independent evolutionary origins, respectively, while the latter was subdivided into CCD1, CCD4, CCD-like, and NCED, which may have come from a common ancestor. In addition, the results of RNA-seq showed that the expression patterns of pepper CaCCO genes were different in the tissues tested, and only few genes were expressed at high levels such as CaCCD1a, CaCCD4a, CaNCED3, and CaCCD1b. For hydrogen peroxide (H2O2) and other abiotic stresses, such as plant hormones, heat, cold, drought, and NaCl treatments, induction of about half of the CaCCO genes was observed. Moreover, the expression patterns of CaCCOs were further investigated under heat, cold, drought, and NaCl treatments using quantitative real-time PCR (qRT-PCR), and most members were responsive to these stresses, especially some CaCCOs with significant expression changes were identified, such as CaCCD4c, CaCCD-like1, CaCCD8, and CaCCD1b, suggesting the important roles of CaCCOs in abiotic stress responses. All these results will provide a valuable analytical basis for understanding the evolution and functions of the CCO family in plants.

The Hsp70 Gene Family in Solanum tuberosum: Genome-Wide Identification, Phylogeny, and Expression Patterns.

Heat shock protein 70 (Hsp70) family members play important roles in protecting plants against abiotic stresses, including salt, drought, heat, and cold. In this study, 20 putative StHsp70 genes were identified in potato (Solanum tuberosum L.) through the integration of the gene structures, chromosome locations, phylogenetic relationships, and expression profiles. These StHsp70 genes were classified into five sub-families based on phylogenetic analysis. Chromosome mapping revealed that they were unevenly and unequally distributed on 10 of the 12 chromosomes. Furthermore, segmental and tandem duplication events contributed to the expansion of the StHsp70 genes. Phylogenetic tree of the HSP70 genes from potato and other plant species revealed multiple sub-families. These findings indicated a common ancestor which had generated diverse sub-families prior to a mono-dicot split. In addition, expression analysis using RNA-seq revealed that the majority of these genes were expressed in at least one of the tested tissue, and were induced by Phytophthora infestans. Then, based on qRT-PCR analysis, the results showed that the transcript levels of some of the StHsp70 genes could be remarkably induced by such abiotic and hormone stresses, which indicated their potential roles in mediating the responses of potato plants to both abiotic and biotic stress conditions.

Characterization of NoV P particle-based chimeric protein vaccines developed from two different expression systems.

The Norovirus (NoV) P domain, with three surface loops for foreign antigen insertion, has been demonstrated as an excellent platform for antigen presentation and novel vaccine development. The P domain alone can self-assemble into a P dimer, 12-mer small particle or 24-mer P particle, and vaccines based on those particles may elicit different levels of immunogenicity. Currently, P particles are generally produced in soluble expression systems in Escherichia coli, mainly in the 24-mer form. However, the low yield of the soluble protein has hindered further clinical applications of P particle-based protein vaccines. In this study, we inserted the Alzheimer's disease (AD) immunogen Aß1-6 into the three loops of the P particle to generate an AD protein vaccine. To increase the yield of this chimeric protein, we tested the generation of proteins in a soluble expression system and an inclusion body expression system separately in E. coli. The result showed that the inclusion body expression system could greatly enhance the product yield of the chimeric protein compared with the soluble expression system. The refolded protein from the inclusion bodies was mainly in the 12-mer form, while the protein generated from the soluble supernatant was mainly in the 24-mer form. Moreover, the immunogenicity of soluble proteins was significantly stronger than that of the refolded proteins. Thus, comparisons between the two expression methods suggested that the soluble expression system generated chimeric P particles with better immunogenicity, while inclusion body expression system yielded more P particle proteins.

Establishment of a novel method without sequence modification for developing NoV P particle-based chimeric vaccines.

The Norovirus (NoV) P particle (PP) is a subviral particle formed by 24 copies of the protruding (P) domain of the capsid protein. Each P domain has three surface loops that can be used for foreign antigen presentation. Hence, PPs have been demonstrated to be an excellent platform for vaccine development against many pathogens. However, current processes for preparing those chimeric PP vaccines vary and would change the original sequence of the PP. A detailed strategy also has not been reported for inserting a foreign antigen into all three loops. In order to develop a novel method for preparing distinct types of PP-based protein vaccines, we created two restriction enzyme sites (EagI and KpnI) in the P domain by site-directed mutagenesis without changing its original sequence. A synthesized gene with three copies of the Alzheimer's disease (AD) immunogen Aß1-6 was then incorporated in loop2 of the P domain. Additionally, a synthesized gene with one copy of Aß1-6 was inserted into each loop of the P domain. Furthermore, two recombinant proteins PP-3 copy-Aß1-6-loop2 and PP-1 copy-Aß1-6-loop123 were successfully purified without affecting PP formation. Particle size analysis and TEM observations demonstrated that the two chimeric P particles were still able to form 24-mer nanoparticles. Moreover, the two chimeric PP-based AD vaccines could both efficiently elicit strong immune responses in the mouse model. In conclusion, we have successfully established a novel method for preparing vaccines based on the NoV PP which would not affect PP sequence and function.

Large-scale purification of pharmaceutical-grade plasmid DNA using tangential flow filtration and multi-step chromatography.

The demand for pharmaceutical-grade plasmid DNA in vaccine applications and gene therapy has been increasing in recent years. In the present study, a process consisting of alkaline lysis, tangential flow filtration, purification by anion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography was developed. The final product met the requirements for pharmaceutical-grade plasmid DNA. The chromosomal DNA content was <1 µg/mg plasmid DNA, and RNA was not detectable by agarose gel electrophoresis. Moreover, the protein content was <2 µg/mg plasmid DNA, and the endotoxin content was <10 EU/mg plasmid DNA. The process was scaled up to yield 800 mg of pharmaceutical-grade plasmid DNA from approximately 2 kg of bacterial cell paste. The overall yield of the final plasmid DNA reached 48%. Therefore, we have established a rapid and efficient production process for pharmaceutical-grade plasmid DNA.