Juan Huang

Association mapping is a powerful approach for dissecting the genetic architecture of complex quantitative traits using high-density SNP markers in maize. Here, we expanded our association panel size from 368 to 513 inbred lines with 0.5 million high quality SNPs using a two-step data-imputation method which combines identity by descent (IBD) based projection and k-nearest neighbor (KNN) algorithm. Genome-wide association studies (GWAS) were carried out for 17 agronomic traits with a panel of 513 inbred lines applying both mixed linear model (MLM) and a new method, the Anderson-Darling (A-D) test. Ten loci for five traits were identified using the MLM method at the Bonferroni-corrected threshold -log10 (P) >5.74 (α=1). Many loci ranging from one to 34 loci (107 loci for plant height) were identified for 17 traits using the A-D test at the Bonferroni-corrected threshold -log10 (P) >7.05 (α=0.05) using 556809 SNPs. Many known loci and new candidate loci were only observed by the A-D test, a few of which were also detected in independent linkage analysis. This study indicates that combining IBD based projection and KNN algorithm is an efficient imputation method for inferring large missing genotype segments. In addition, we showed that the A-D test is a useful complement for GWAS analysis of complex quantitative traits. Especially for traits with abnormal phenotype distribution, controlled by moderate effect loci or rare variations, the A-D test balances false positives and statistical power. The candidate SNPs and associated genes also provide a rich resource for maize genetics and breeding.

Maize is one of the most important crops globally, and it shows remarkable genetic diversity. Knowledge of this diversity could help in crop improvement; however, gold-standard genomes have been elucidated only for modern temperate varieties. Here, we present a high-quality reference genome (contig N50 of 15.78 megabases) of the maize small-kernel inbred line, which is derived from a tropical landrace. Using haplotype maps derived from B73, Mo17 and SK, we identified 80,614 polymorphic structural variants across 521 diverse lines. Approximately 22% of these variants could not be detected by traditional single-nucleotide-polymorphism-based approaches, and some of them could affect gene expression and trait performance. To illustrate the utility of the diverse SK line, we used it to perform map-based cloning of a major effect quantitative trait locus controlling kernel weight-a key trait selected during maize improvement. The underlying candidate gene ZmBARELY ANY MERISTEM1d provides a target for increasing crop yields.

A new anthrax vaccine under clinical investigation is based on recombinant Bacillus anthracis protective antigen (rPA). Here, we investigated microneedle-based cutaneous and nasal mucosal delivery of rPA in mice and rabbits. In mice, intradermal (id) delivery achieved up to 90% seroconversion after a single dose, compared with 20% after intramuscular (im) injection. Intranasal (inl) delivery of a liquid formulation required 3 doses to achieve responses that were comparable with those achieved via the id or im routes. In rabbits, id delivery provided complete protection against aerosol challenge with anthrax spores; in addition, novel powder formulations administered inl provided complete protection, whereas a liquid formulation provided only partial protection. These results demonstrate, for the first time, that cutaneous or nasal mucosal administration of rPA provides complete protection against inhalational anthrax in rabbits. The novel vaccine/device combinations described here have the potential to improve the efficacy of rPA and other biodefense vaccines.

Kernel row number (KRN) is an important component of yield during the domestication and improvement of maize and controlled by quantitative trait loci (QTL). Here, we fine-mapped a major KRN QTL, KRN4, which can enhance grain productivity by increasing KRN per ear. We found that a ~3-Kb intergenic region about 60 Kb downstream from the SBP-box gene Unbranched3 (UB3) was responsible for quantitative variation in KRN by regulating the level of UB3 expression. Within the 3-Kb region, the 1.2-Kb Presence-Absence variant was found to be strongly associated with quantitative variation in KRN in diverse maize inbred lines, and our results suggest that this 1.2-Kb transposon-containing insertion is likely responsible for increased KRN. A previously identified A/G SNP (S35, also known as Ser220Asn) in UB3 was also found to be significantly associated with KRN in our association-mapping panel. Although no visible genetic effect of S35 alone could be detected in our linkage mapping population, it was found to genetically interact with the 1.2-Kb PAV to modulate KRN. The KRN4 was under strong selection during maize domestication and the favorable allele for the 1.2-Kb PAV and S35 has been significantly enriched in modern maize improvement process. The favorable haplotype (Hap1) of 1.2-Kb-PAV-S35 was selected during temperate maize improvement, but is still rare in tropical and subtropical maize germplasm. The dissection of the KRN4 locus improves our understanding of the genetic basis of quantitative variation in complex traits in maize.

Maize (Zea mays) is a major staple crop. Maize kernel size and weight are important contributors to its yield. Here, we measured kernel length, kernel width, kernel thickness, hundred kernel weight, and kernel test weight in 10 recombinant inbred line populations and dissected their genetic architecture using three statistical models. In total, 729 quantitative trait loci (QTLs) were identified, many of which were identified in all three models, including 22 major QTLs that each can explain more than 10% of phenotypic variation. To provide candidate genes for these QTLs, we identified 30 maize genes that are orthologs of 18 rice (Oryza sativa) genes reported to affect rice seed size or weight. Interestingly, 24 of these 30 genes are located in the identified QTLs or within 1 Mb of the significant single-nucleotide polymorphisms. We further confirmed the effects of five genes on maize kernel size/weight in an independent association mapping panel with 540 lines by candidate gene association analysis. Lastly, the function of ZmINCW1, a homolog of rice GRAIN INCOMPLETE FILLING1 that affects seed size and weight, was characterized in detail. ZmINCW1 is close to QTL peaks for kernel size/weight (less than 1 Mb) and contains significant single-nucleotide polymorphisms affecting kernel size/weight in the association panel. Overexpression of this gene can rescue the reduced weight of the Arabidopsis (Arabidopsis thaliana) homozygous mutant line in the AtcwINV2 gene (Arabidopsis ortholog of ZmINCW1). These results indicate that the molecular mechanisms affecting seed development are conserved in maize, rice, and possibly Arabidopsis.

A simple and effective method was established for separation and characterization of flavonoid constituents in Radix Astragali (RA) by combination of ultra-high-pressure liquid chromatography with LTQ-Orbitrap tandem mass spectrometry (u-HPLC-LTQ-Orbitrap-MS(n)). For three major structural types of flavonoids, the proposed fragmentation pathways and major diagnostic fragment ions of isoflavones, pterocarpans and isoflavans were investigated to trace isoflavonoid derivatives in crude plant extracts. Based on the systematic identification strategy, 48 constituents were rapidly detected and characterized or tentatively identified, many of which were first reported in RA. The u-PHLC-LTQ-Orbitrap MS(n) platform was proved as an effective tool for rapid qualitative analysis of secondary metabolite productions from natural resources.

Low temperature is a limiting factor of rice productivity and geographical distribution. Wild rice (Oryza rufipogon Griff.) is an important germplasm resource for rice improvement. It has superior tolerance to many abiotic stresses, including cold stress, but little is known about the mechanism underlying its resistance to cold.This study elucidated the molecular genetic mechanisms of wild rice in tolerating low temperature. Comprehensive transcriptome profiles of two rice genotypes (cold-sensitive ce 253 and cold-tolerant Y12-4) at the germinating stage under cold stress were comparatively analyzed. A total of 42.44-68.71 million readings were obtained, resulting in the alignment of 29,128 and 30,131 genes in genotypes 253 and Y12-4, respectively. Many common and differentially expressed genes (DEGs) were analyzed in the cold-sensitive and cold-tolerant genotypes. Results showed more upregulated DEGs in the cold-tolerant genotype than in the cold-sensitive genotype at four stages under cold stress. Gene ontology enrichment analyses based on cellular process, metabolic process, response stimulus, membrane part, and catalytic activity indicated more upregulated genes than downregulated ones in the cold-tolerant genotype than in the cold-sensitive genotype. Quantitative real-time polymerase chain reaction was performed on seven randomly selected DEGs to confirm the RNA Sequencing (RNA-seq) data. These genes showed similar expression patterns corresponding with the RNA-Seq method. Weighted gene co-expression network analysis (WGCNA) revealed Y12-4 showed more positive genes than 253 under cold stress. We also explored the cold tolerance gene LTG5 (Low Temperature Growth 5) encoding a UDP-glucosyltransferase. The overexpression of the LTG5 gene conferred cold tolerance to indica rice.Gene resources related to cold stress from wild rice can be valuable for improving the cold tolerance of crops.

Honeysuckle (Lonicera japonica Thunb) is a traditional Chinese medicine (TCM) with an antipathogenic activity. MicroRNAs (miRNAs) are small non-coding RNA molecules that are ubiquitously expressed in cells. Endogenous miRNA may function as an innate response to block pathogen invasion. The miRNA expression profiles of both mice and humans after the ingestion of honeysuckle were obtained. Fifteen overexpressed miRNAs overlapped and were predicted to be capable of targeting three viruses: dengue virus (DENV), enterovirus 71 (EV71) and SARS-CoV-2. Among them, let-7a was examined to be capable of targeting the EV71 RNA genome by reporter assay and Western blotting. Moreover, honeysuckle-induced let-7a suppression of EV71 RNA and protein expression as well as viral replication were investigated both in vitro and in vivo. We demonstrated that let-7a targeted EV71 at the predicted sequences using luciferase reporter plasmids as well as two infectious replicons (pMP4-y-5 and pTOPO-4643). The suppression of EV71 replication and viral load was demonstrated in two cell lines by luciferase activity, RT-PCR, real-time PCR, Western blotting and plaque assay. Furthermore, EV71-infected suckling mice fed honeysuckle extract or inoculated with let-7a showed decreased clinical scores and a prolonged survival time accompanied with decreased viral RNA, protein expression and virus titer. The ingestion of honeysuckle attenuates EV71 replication and related pathogenesis partially through the upregulation of let-7a expression both in vitro and in vivo. Our previous report and the current findings imply that both honeysuckle and upregulated let-7a can execute a suppressive function against the replication of DENV and EV71. Taken together, this evidence indicates that honeysuckle can induce the expression of let-7a and that this miRNA as well as 11 other miRNAs have great potential to prevent and suppress EV71 replication.

Intranasal (IN) vaccination represents an attractive non-invasive alternative to needle-based injection and provides superior protection at mucosal surfaces. However, new formulations are needed to improve efficacy and reduce the refrigerated storage and distribution requirements associated with standard liquid vaccines. Here, we describe a powder formulation of whole inactivated influenza virus and a novel IN delivery platform. The powder-formulated vaccine elicited a significant serum antibody response in rats that was at least as strong as that provided by the liquid vaccine administered IN or via intramuscular (IM) injection. Significant nasal IgA responses were also observed solely after IN delivery. This study demonstrates for the first time the generation of potent nasal mucosal and systemic immune responses using an IN delivered influenza vaccine powder and suggests an alternative approach to vaccination against influenza and other infectious diseases.

Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6–8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7–8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association Anthrax remains a serious threat worldwide as a bioterror agent. A second-generation anthrax vaccine currently under clinical evaluation consists of a recombinant Protective Antigen (rPA) of Bacillus anthracis. We have previously demonstrated that complete protection against inhalational anthrax can be achieved in a rabbit model, by intranasal delivery of a powder rPA formulation. Here we describe the preformulation and formulation development of such powder formulations. The physical stability of rPA was studied in solution as a function of pH and temperature using circular dichroism (CD), and UV-visible absorption and fluorescence spectroscopies. Extensive aggregation of rPA was observed at physiological temperatures. An empirical phase diagram, constructed using a combination of CD and fluorescence data, suggests that rPA is most thermally stable within the pH range of 6–8. To identify potential stabilizers, a library of GRAS excipients was screened using an aggregation sensitive turbidity assay, CD, and fluorescence. Based on these stability profiles, spray freeze-dried (SFD) formulations were prepared at pH 7–8 using trehalose as stabilizer and a CpG-containing oligonucleotide adjuvant. SFD formulations displayed substantial improvement in storage stability over liquid formulations. In combination with noninvasive intranasal delivery, such powder formulations may offer an attractive approach for mass biodefense immunization. © 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Maize seedlings are very sensitive to chilling, especially during the transition phase from heterotrophic to autotrophic growth. Genetic dissection of the genetic basis of chilling tolerance would provide useful information for genetic improvement of maize inbreds. In this study, genome-wide association analysis was conducted to explore the genetic architecture of maize chilling tolerance at the seed germination and seedling stages with an association panel of 125 inbreds. Ten tolerance indices (ratios of the performance of 10 germination rates and seedling growth-related traits under chilling stress and control conditions) were investigated to assess the ability of chilling tolerance of the inbreds, and a total of 43 single nucleotide polymorphisms associated with chilling tolerance were detected, with none of them being related to chilling tolerance at both the germination and seedling stages simultaneously. Correlation analysis also revealed that the genetic basis of chilling tolerance at the seed germination stage is generally different from that at the seedling stage. In addition, a total of 40 candidate genes involving 31 of the 43 single nucleotide polymorphisms were predicted, and were grouped into five categories according to their functions. The possible roles of these candidate genes in chilling tolerance were also discussed.

Both grain size and grain number are significant for rice yield. In the past decade, a number of genes related to grain size and grain number have been documented, however, the regulatory mechanisms underlying them remains ambiguous.We identified a rice small grain (sg2) mutant in an EMS mutant library generated from an indica variety, Shuhui498. Using the MutMap gene mapping strategy, we identified two linkage regions on chromosome 7 and 8, respectively, consistent with the segregation ratios in the F2 population. We focused on the linkage region on chromosome 8, and named this locus as 08sg2. One of three SNPs identified in the linkage region was located in an exon of OsBAK1, leading to a nonsynonymous mutation in the kinase domain. The plant harboring the mutant version 08sg2 locus exhibited a decreased grain size, grain number and plant height. Cytological analysis indicated that 08SG2 regulated spikelet hull development by affecting cell proliferation. The grain size and number of knockout mutants of OsBAK1 in the japonica background were significantly decreased, but less so than in 08sg2, supporting the idea that the SNP in OsBAK1 was responsible for the 08sg2 phenotype, but that 08SG2/OsBAK1 function differently in indica and japonica backgrounds. 08sg2 was insensitive to 24-epiBL, and the expression of BR-related genes was obviously altered in 08sg2. The proportionally decreased grain length when 08sg2 and GS3 were combined indicate that 08SG2 and GS3 regulate grain length independently.Our work shows that 08SG2/OsBAK1 is important for rice yield in both indica and japonica backgrounds, by regulating grain size and grain number, and the function of 08SG2/OsBAK1 is obviously affected by genetic background. The amino acid substituted in 08sg2 is highly conserved among different species, supporting the idea that it is important for the molecular function of 08SG2/OsBAK1. Together, our work is helpful for fully understanding the function of 08SG2/OsBAK1.

In maize (Zea mays), kernel weight is an important component of yield that has been selected during domestication. Many genes associated with kernel weight have been identified through mutant analysis. Most are involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, few quantitative trait loci (QTLs) underlying quantitative variation in kernel weight have been cloned. Here, we characterize a QTL, qKW9, associated with maize kernel weight. This QTL encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of ndhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qkw9 background, C-to-U editing of ndhB was abolished, and photosynthesis was reduced, resulting in less maternal photosynthate available for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis for maize grain yield production.

Abstract Grain size is one of the major traits that determine rice grain yield and quality. The GS3 gene is the first major quantitative trait locus (QTL) that was identified in regulating rice grain length and weight. It was reported that the gs3 allele with a mutation in the organ size regulation (OSR) domain of the GS3 protein produced longer grains. In this study, we used the CRISPR/Cas9 gene editing technology to introduce an edited gs3 allele into our indica maintainer line, Mei1B, to enhance its grain yield and quality. Through molecular analysis and sequencing, a homologous edited- gs3 mutant line without any transgene was obtained in the T 1 generation and was named Mei2B. A superior male sterile line Mei2A was generated by backcrossing the cytoplasmic male sterile (CMS) line Mei1A with Mei2B. Mei2B had a higher grain quality and yield compared to its wild-type Mei1B. Its grain length increased by 7.9%, its length/width ratio increased from 3.89 to 4.19, TGW increased by 6.7%, and grain yield per plant increased by 14.9%. In addition, genetic improvement of other quality traits including brown rice length (6.83 mm), brown rice grain length/width ratio (3.61), matched the appearance standards set for traditional Simiao (silk seedling) type cultivars. Two restorer lines were outcrossed to both Mei1A and Mei2A to produce hybrid rice. Compared to two hybrids of Mei1A, the hybrids of Mei2A had longer grains, higher length/width ratio, TGW, and yield per plant. In addition, the hybrids of Mei2A showed a better grain appearance including better translucency, a lower chalky rice rate, and degree of chalkiness than the hybrids of Mei1A. These results demonstrated that the introduction of an elite gs3 allele into Mei1A via CRISPR/Cas9 gene editing technology led to significant genetic improvement of the rice grain. The resultant CMS line Mei2A( gs3 ) displayed much higher grain quality and yield than the original Mei1A. Therefore, our study demonstrated that the targeted genetic improvement via gene editing technology can enhance rice breeding, especially the breeding of three-line hybrid rice.

Heat shock transcription factors (Hsfs), which act as important transcriptional regulatory proteins, play crucial roles in plant developmental processes, and stress responses. Recently, the genome of the shrub willow Salix suchowensis was fully sequenced. In this study, a total of 27 non-redundant Hsf genes were identified from the S. suchowensis genome. Phylogenetic analysis revealed that the members of the SsuHsf family can be divided into three groups (class A, B, and C) based on their structural characteristics. Promoter analysis indicated that the SsuHsfs promoters included various cis-acting elements related to hormone and/or stress responses. Furthermore, the expression profiles of 27 SsuHsfs were analyzed in different tissues and under various stresses (heat, drought, salt, and ABA treatment) using RT-PCR. The results demonstrated that the SsuHsfs were involved in abiotic stress responses. Our results contribute to a better understanding of the complexity of the SsuHsf gene family, and will facilitate functional characterization in future studies.

OBJECTIVE: To study the clinical features of children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: A retrospective analysis was performed for the clinical data of 13 children with SARS-CoV-2 infection who hospitalized in a Changsha hospital. RESULTS: All 13 children had the disease onset due to family aggregation. Of the 13 children, 2 had no symptoms, and the other 11 children had the clinical manifestations of fever, cough, pharyngeal discomfort, abdominal pain, diarrhea, convulsions, or vomiting. As for clinical typing, 7 had mild type, 5 had common type, and 1 had severe type. The median duration of fever was 2 days in 6 children. All 13 children had normal levels of peripheral blood lymphocyte counts, immunoglobulins, CD4, CD8, and interleukin-6. The median time to clearance of SARS-CoV-2 was 13 days in the nasopharyngeal swabs of the 13 children. Three children presented false negatives for RT-PCR of SARS-CoV-2. SARS-CoV-2 RNA remained detectable in stools for 12 days after the nasopharyngeal swab test yielded a negative result. Abnormal CT findings were observed in 6 children. All 13 children were cured and discharged and they were normal at 2 weeks after discharge. CONCLUSIONS: Intra-family contact is the main transmission route of SARS-CoV-2 infection in children, and there is also a possibility of fecal-oral transmission. Mild and common types are the major clinical types in children with SARS-CoV-2 infection, and cytokine storm is not observed. Children with SARS-CoV-2 infection tend to have a good short-term prognosis, and follow-up is needed to observe their long-term prognosis. Multiple nucleic acid tests should be performed for patients with SARS-CoV-2 infection and their close contacts by multiple site sampling.

Southern Chinese Medicine (SCM) is an important sect of Traditional Chinese Medicine (TCM) with its own special cultural style. Species identification is essential for TCM quality control because authentic herbs are possibly substituted with adulterants that would threaten the health of the public or even cause death. Here, we provided the first local reference DNA barcode library based on the second internal transcribed spacer (ITS2) for the molecular identification of SCM. A total of 1512 specimens of southern herbs representing 359 species were collected under the instructions and identification of taxonomic experts. Genomic DNA was extracted, and the PCR reaction proceeded according to standard procedures. After Sanger sequencing, sequence assembling and annotation, a reliable ITS2 barcode library with 1276 sequences from 309 species of Southern herbs was constructed. The PCR efficiency of the whole samples was 84.39%. Characteristics of the ITS2 barcode were analyzed, including sequence lengths and GC contents in different taxa. Neighbor-joining trees based on Kimura 2-Parameter (K2P) genetic distances showed a 67.56% successful rate of species identification with ITS2 barcode. In addition, 96.57% of species could be successfully identified at the genus level by the BLAST method. Eleven plant species were discovered to be cryptic. In addition, we found that there is an incorrect sequence existing in the public database, making a reliable local DNA barcode reference more meaningful. ITS2 barcodes exhibit advantages in TCM identification. This DNA barcode reference library could be used in Southern Chinese Medicine quality control, thus contributing to protecting public health.

CRP (Citri Reticulatae Pericarpium), a famous traditional Chinese medicine, has also been extensively used in foods and condiments in dietary practice for centuries. According to the Chinese Pharmacopeia (2015 edition) it contains two subtypes, Guangchenpi (GCP) and Chenpi (CP). GCP exclusively originates from the pericarp of Citrus reticulata 'Chachi' cultivar and it's generally believed that GCP has superior qualities compared with the other main cultivars (CP). In the present study, an integrated approach combining LC-QTOF MS-based untargeted metabolomics analysis and DNA barcoding molecular identification was conducted to study the genetic diversity and chemical differences between GCP and CP. A validated UPLC-QTOF MS metabolomics method was established to identify markers by using PCA and OPLS-DA models. 34 identified metabolites could be used as chemical markers to distinguish effectively between the two subtypes. Among them polymethoxyflavones (PMF) such as hexamethoxyflavone (nobiletin and natsudaidain), pentamethoxyflavone (tangeretin and sinensetin), and tetramethoxyflavone are the most influential markers. Support vector machines were employed to classify all the samples and these markers showed good prediction accuracy (100%). The results of DNA barcoding showed that the secondary structure of the ITS2 sequences were significantly different among GCP and other three cultivars. The study indicated the integrated method could be a powerful and reliable analytical tool for differentiating GCP from CP.

Species in genus Amomum always have important medicinal and economic values. Classification of Amomum using morphological characters has long been a challenge because they exhibit high similarity. The main goals of this study were to mine genetic markers from cp genomes for Amomum species identification and discover their evolutionary history through comparative analysis.Three species Amomum villosum, Amomum maximum and Amomum longipetiolatum were sequenced and annotated for the complete chloroplast (cp) genomes, and the cp genomes of A. longipetiolatum and A. maximum were the first reported. Three cp genomes exhibited typical quadripartite structures with 163,269-163,591 bp in length. Each genome encodes 130 functional genes including 79 protein-coding, 26 tRNAs and 3 rRNAs genes. 113-152 SSRs and 99 long repeats were identified in the three cp genomes. By designing specific primers, we amplified the highly variable loci and the mined genetic marker ccsA exhibited a relatively high species identification resolution in Amomum. The nonsynonymous and synonymous substitution ratios (Ka/Ks) in Amomum and Alpinia showed that most genes were subjected to a purifying selection. Phylogenetic analysis revealed the evolutionary relationships of Amomum and Alpinia species and proved that Amomum is paraphyletic. In addition, the sequenced sample of A. villosum was found to be a hybrid, becoming the first report of natural hybridization of this genus. Meanwhile, the high-throughput sequencing-based ITS2 analysis was proved to be an efficient tool for interspecific hybrid identification and with the help of the chloroplast genome, the hybrid parents can be also be determined.The comparative analysis and mined genetic markers of cp genomes were conducive to species identification and evolutionary relationships of Amomum.

This paper reviews the developments in noninvasive methods of drug delivery, with a focus on the delivery of vaccines via the respiratory tract. Recent results indicate that the respiratory system, and the nasal mucosa in particular, provide a valuable target site for immunisation against respiratory and mucosal pathogens. Vaccine delivery via the nasal and pulmonary routes each present distinct sets of performance requirements. Current delivery systems in development for both routes are reviewed herein. The storage and respiratory delivery of drugs and vaccines in powder form has been shown to provide improved stability and extended retention time in the respiratory mucosa. These features, in addition to the noninvasive nature of respiratory delivery, can provide benefits to public health vaccination campaigns, facilitating mass vaccination without the high cost of maintaining cold-chain storage.

The purpose of this research was to prepare a dry powder vaccine formulation containing whole inactivated influenza virus (VIIV) and a mucoadhesive compound suitable for nasal delivery. Powders containing WIIV and either lactose or trehalose were produced by lyophilization. A micro-ball mill was used to reduce the lyophilized cake to sizes suitable for nasal delivery. Chitosan flakes were reduced in size using a cryo-milling technique. Milled powders were sieved between 45 and 125 μm aggregate sizes and characterized for particle size and distribution, morphology, and flow properties. Powders were blended in the micro-ball mill without the ball. Lyophilization followed by milling produced irregularly shaped, polydisperse particles with a median primary particle diameter of ≈21 μm and a yield of ≈37% of particles in the 45 to 125 μm particle size range. Flow properties of lactose and trehalose powders after lyophilization followed by milling and sieving were similar. Cryo-milling produced a small yield of particles in the desired size range (<10%). Lyophilization followed by milling and sieving produced particles suitable for nasal delivery with different physicochemical properties as a function of processing conditions and components of the formulation. Further optimization of particle size and morphology is required for these powders to be suitable for clinical evaluation.

Porcine reproductive and respiratory syndrome virus (PRRSV) has been mainly responsible for the heavy economic losses in many swine-producing regions. Current vaccination strategies and antiviral drugs provide only limited protection. Consequently, there is a need to develop a new antiviral strategy. In this study, two recombinant adenoviruses expressing short-hairpin RNAs (shRNAs) directed against ORF1b of PRRSV S1 strain were constructed and the inhibition of PRRSV replication was determined. The results showed that pretreatment with these shRNAs delivered by recombinant adenovirus could induce a significant inhibition of viral RNA and protein level in Marc-145 cells infected with PRRSV S1 strains. One recombinant adenovirus (rAd-P2) was found to be also effective in inhibiting the replication of highly virulent PRRSV SY0608 strain in Marc-145 cells and porcine alveolar macrophages at both the protein and ORF1b mRNA level. The antiviral effect was dose-dependent and sustained for at least 96h. Twenty 6-week old piglets were assigned to four groups each with five piglets. Groups 1 and 2 were inoculated intramuscularly with rAd-P2 and mock construct rAd-mP2 individually. After 24h, groups 1, 2 and 3 were challenged intramuscularly with the SY0608 strain. Group 4 remained unchallenged but with PBS as mock. The results showed that the viral load of PRRSV in serum and lung tissue of swine was suppressed effectively by rAd-P2. The clinical signs and pathological lesions in the pigs inoculated with rAd-P2 were milder than those in rAd-mP2 negative and PRRSV control. These results indicated that shRNAs mediated by the adenovirus could inhibit PRRSV infection sufficiently in vitro as well as in vivo. RNAi mediated by recombinant adenovirus might be a potential new tool for controlling PRRSV infection. Of course, the protective efficiency of rAd-P2 should be made by using a large number of pigs in future.

The lateral root of Aconitum carmichaeli, a popular traditional Chinese medicine, has been widely used to treat rheumatic diseases. For decades, diterpenoid alkaloids have dominated the phytochemical and biomedical research on this plant. In this study, a rapid and sensitive method based on ultra high performance liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry was developed to characterize the diterpenoid alkaloids in Aconitum carmichaeli. Based on an optimized chromatographic condition, more than 120 diterpenoid alkaloids were separated with good resolution. Using a systematic strategy that combines high resolution separation, highly accurate mass measurements and a good understanding of the diagnostic fragment-based fragmentation patterns, these diterpenoid alkaloids were identified or tentatively identified. The identification of these chemicals provided essential data for further phytochemical studies and toxicity research of Aconitum carmichaeli. Moreover, the ultra high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry platform was an effective and accurate tool for rapid qualitative analysis of secondary metabolite productions from natural resources.

Bupi Yishen Formula, a Chinese medicine preparation, has been clinically applied for the recovery of chronic kidney disease and for delaying its progress. Nevertheless, the chemical components in Bupi Yishen Formula have yet to be fully clarified. Ultra-high performance liquid chromatography with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MSn) and triple-quadrupole tandem mass spectrometry (UHPLC-TQ-MS/MS) methods were developed for qualitative chemical profiling and multi-components quantitative analysis in Bupi Yishen Formula. The chromatographic separation was performed on a Phenomenex Kinetex C18 column (2.1×100 mm i.d., 1.7 μm) using gradient elution of water (A) and acetonitrile (B) both containing 0.1% formic acid. Eighty-six compounds, including flavones, saponins, phenolic acids and other compounds were authenticated or temporarily deduced according to their retention behaviors, mass mensuration and characteristic fragment ions with those elucidated references or literature. Among the herbal medicinal materials of the formula, Astragali Radix, Codonopsis Radix, Salviae Miltiorrhizae Radix Rhizoma and Polygoni Multiflori Radix Praeparata contributed to the bulk of the dissolved metabolites of the formula extraction. In addition, seven analytes were simultaneously determined by UHPLC-TQ-MS/MS, which was validated and has managed to determine major components in Bupi Yishen Formula. The study indicated that the established qualitative and quantitative methods would be potent and dependable analytical tools for characterizing multi-constituent in complex prescriptions Decoction and provided a basis for the evaluation of bioactive components in Bupi Yishen Formula.

The anthocyanin biosynthesis of rice is a major concern due to the potential nutritional value. Purple appears in various organs and tissues of rice such as pericarp, flower organs, leaves, leaf sheaths, internodes, ligules, apex, and stigma. At present, there are many studies on the color of rice pericarp, but the gene and mechanism of other organs such as leaves are still unclear, and the gene regulatory network of specific organ coloring has not been systematically understood. In this study, genetic analysis demonstrated that the purple leaf traits of rice were regulated by a recessive gene. The green leaf cultivar Y58S and purple leaf cultivar XianHongB were used to construct the mapping population. A set of near isogenicline (NIL) (BC3F1) was bred via crossing and back-crossing. The generations of BC3F2 appeared to separate four phenotypes, pl1, pl2, pl3, and pl4, due to the occurrence of a purple color in different organs. We constructed three bulked segregant analysis (BSA) pools (pl1-pl2, pl1-pl3, and pl1-pl4) by using the separated generations of BC3F5 and mapped the purple leaf gene plr4 to the vicinity of 27.9-31.1 Mb on chromosome 4. Subsequently, transcriptome sequencing (RNA-Seq) for pl3 and pl2 was used to analyze the differentially expressed genes in the localization interval, where 12 unigenes exhibited differential expression in which two genes (Os04g0577800, Os04g0616400) were downregulated. The two downregulated genes (Os04g0577800 and Os04g0616400) are possible candidate genes because of the recessive genetic characteristics of the purple leaf genes. These results will facilitate the cloning of plr4 and illustrate the molecular mechanisms of the anthocyanin synthesis pathway.

Gastrodin, a sedative drug, is a highly water-soluble phenolic glucoside with poor liposolubility but exhibits good oral bioavailability. The current study aims to investigate whether glucose transporters (GLTs) are involved in the intestinal absorption of gastrodin. The intestinal absorption kinetics of gastrodin was determined using the rat everted gut sac model, the Caco-2 cell culture model and the perfused rat intestinal model. In vivo pharmacokinetic studies using diabetic rats with high GLT expression were performed. Saturable intestinal absorption of gastrodin was observed in rat everted gut sacs. The apparent permeability (Papp) of gastrodin from the apical (A) to basolateral (B) side in Caco-2 cells was two-fold higher than that from B to A. Glucose or phlorizin, a sodium-dependent GLT (SGLT) inhibitor, reduced the absorption rates of gastrodin from perfused rat intestines. In vivo pharmacokinetic studies showed that the time of maximum plasma gastrodin concentration (Tmax) was prolonged from 28 to 72 min when orally co-administered with four times higher dose of glucose. However, the Tmax of gastrodin in diabetic rats was significantly lowered to 20 min because of the high intestinal SGLT1 level. In conclusion, our findings indicate that SGLT1 can facilitate the intestinal absorption of gastrodin.

The raw and processed roots of Plygonum multiflorum Thunb (PM) are used to treat different diseases in clinical practice. In order to clarify the influence of processing, a comparative study of chemical substance analysis was carried out. As the xenobiotics with a high enough exposure in target organs being considered as the potential effective or toxicity components, an in vivo study was also implemented to characterize the constitutes and metabolites, and meanwhile, the factor of compatibility with black bean were also considered. As a result, a total of 148 compounds were detected in PM extracts and more than 40 compounds were only detected in the processed products, which were probably new components produced during the steaming process. In in vivo study, 7 prototype components and 66 metabolites were detected or tentatively identified, 24 of which were reported for the first time. Our results indicated that processing greatly changed the chemical composition of PM and influenced the disposition of the compounds in vivo. To the best of our knowledge, this was the first global comparative study of raw and processed PM. These results expanded our knowledge about the influence of processing of PM and provided the essential data for further efficacy or toxicity studies.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. The current vaccines cannot provide highly effective protection. In this study, the ability of specific short hairpin RNA directed against different genomic regions of PRRSV to inhibit virus replication in MARC-145 cells was examined. Seven plasmids expressing shRNA targeted to GP5 and nucleocapsid (N) protein coding region of PRRSV S1 strain RNA were constructed and delivered into MARC-145 cells. After infection, these cells, transfected with plasmids pSUPER-N3 or pSUPER-G1, showed a significant decrease in virus yield when compared to control cells, by detection using virus titers (TCID50), indirect immunofluorescence assay and real-time RT-PCR. The antiviral effect was sequence-specific and dose-dependent and could sustain for 96 h. Furthermore, by combination of treatment with plasmid pSUPER-N3 and pSUPER-G1, the viral inhibition cloud be significantly increased. In addition, the viral suppression efficiency by shRNA in previously infected cells was not significant different from that induced by shRNA before viral infection. It indicated that administration of the two different shRNA could have a synergistic effect. RNA interference targeting to the various regions of PRRSV might be a potential alternative virus control strategy.

Abstract The ideal plant architecture is a new strategy for super high yield breeding of rice. Tiller angle is an important plant architecture character of rice. A reasonable tiller angle is a key factor for the ideal plant architecture and achieving high-yield breeding. Molecular design breeding is the most potential new direction of crop breeding in the future. The development of accurate and efficient functional molecular markers of target trait genes is crucial for molecular design breeding. The TAC1 ( Tiller Angle Controlling ) gene is the primary gene that regulates tiller angle in rice. This gene can be used to improve the compact plant architecture of indica and japonica rice varieties. The SNP variation from A to G at the fourth intron 3′ splicing point in TAC1 changes plant architecture. Based on the SNP variation, PM-TAC1 was successfully developed as a fluorescent functional molecular marker, via the penta-primer amplification refractory mutation system. Ninety-three rice materials were genotyped using this marker, and the marker was effectively used in rice plant architecture breeding. The successful development of this marker will contribute to the molecular breeding of rice plant architecture.

Emodin (EM) and Emodin-8-O-β-D-glucoside (EG) exists in various medicinal plants such as Rheum palmatum, Aloe vera, Polygonum multiflorum, and Polygonum cuspidatum. They have various pharmacological properties and are considered potential toxic substances. It is necessary to identify the metabolites and distribution in the body. In this study, a comprehensive analytical strategy was developed to characterize the metabolites of EM and EG in vivo (rat plasma, bile, urine, heart, liver, spleen, lung, kidney, and brain) using UHPLC-Q-Exactive Orbitrap MS. As a result, a total of 190 metabolites were identified in the bio-samples, except for the commonly reported glycoside hydrolysis, hydrogenation, hydroxylation, glucuronide conjugation, and sulfate conjugation. We have also discovered new metabolic pathways such as formylation, acetylation, ethylene glycol, lactated, glycerol, malonyl, glyceroyl, hydroxyvaleryl, erythrose, glutaryl, hydroxybenzoyl, glutamyl, hydroxyglutaryl, ascorbyl, aspartylglycine, dihydroxyphenylglycine, trihydroxyphenylglycine, dihydroxymethoxyphenylglycine, and ring-opening emodin. Cluster analysis shows that the EM and EG groups of each sample are clustered together, indicating that the pathway and distribution of EM and EG metabolites are essentially the same. Interestingly, EM was found in all biological samples, and EG was detected in all samples except the bill of EM. The detection of ethylene glycol, glycerol, and glycerate in the liver may be related to the lipid-lowering effects of EM and EG. These results provide valuable information for a deeper understanding of the safety and efficacy of EM and EG, and valuable methods for metabolic characterization.

Introduction: The root of Reynoutria multiflora (Thunb.) Moldenke (RM) has been used widely in formulations of herbal medicines in China for centuries. Raw R. multiflora (RRM) should be processed before use to reduce toxicity and increase efficacy. However, detailed regulation of the processing endpoint is lacking, and the duration of processing can vary considerably. We conducted in-depth research on stilbene glycosides in RM at different processing times. Previously, we discovered that 219 stilbene glycosides changed markedly in quantity and content. Therefore, we proposed that processing causes changes in various chemical groups. Methods: To better explain the mechanism of RM processing for toxicity reduction and efficacy enhancement, we used a method of tandem mass spectrometry described previously to research gallic acid based and catechin based metabolites. Results: A total of 259 metabolites based on gallic acid and 112 metabolites based on catechins were identified. Among these, the peak areas of 157 gallic acid and 81 catechins gradually decreased, those of another 71 gallic acid and 30 catechins first increased and then decreased, those of 14 gallic acid and 1 catechin gradually increased. However, 17 of the gallic acids showed no significant changes. We speculate that many gallic acid metabolites hydrolyze to produce gallic acid; moreover, the dimers/trimers of catechins, after being cleaved into catechins, epicatechin, gallic acid catechins, and epicatechin monomers, are cleaved into gallic acid and protocatechualdehyde under high temperature and high humidity, subsequently participating in the Maillard reaction and browning reactions. Discussion: We showed that processing led to changes in chemical groups, clarification of the groups of secondary metabolites could provide a basis for research on the pharmacological and toxic mechanisms of RM, as well as the screening of related markers.

Rice is one of the most important staple crops in the world; therefore, the improvement of rice holds great significance for enhancing agricultural production and addressing food security challenges. Although there have been numerous studies on the role of single-nucleotide polymorphisms (SNPs) in rice improvement with the development of next-generation sequencing technologies, research on the role of presence/absence variations (PAVs) in the improvement of rice is limited. In particular, there is a scarcity of studies exploring the traits and genes that may be affected by PAVs in rice. Here, we extracted PAVs utilizing resequencing data from 148 improved rice varieties distributed in Asia. We detected a total of 33,220 PAVs and found that the number of variations decreased gradually as the length of the PAVs increased. The number of PAVs was the highest on chromosome 1. Furthermore, we identified a 6 Mb hotspot region on chromosome 11 containing 1091 PAVs in which there were 29 genes related to defense responses. By conducting a genome-wide association study (GWAS) using PAV variation data and phenotypic data for five traits (flowering time, plant height, flag leaf length, flag leaf width, and panicle number) across all materials, we identified 186 significantly associated PAVs involving 20 cloned genes. A haplotype analysis and expression analysis of candidate genes revealed that important genes might be affected by PAVs, such as the flowering time gene OsSFL1 and the flag leaf width gene NAL1. Our work investigated the pattern in PAVs and explored important PAV key functional genes associated with agronomic traits. Consequently, these results provide potential and exploitable genetic resources for rice breeding.

China is rich of germplasm resources of common wild rice (Oryza rufipogon Griff.) and Asian cultivated rice (O. sativa L.) which consists of two subspecies, indica and japonica. Previous studies have shown that China is one of the domestication centers of O. sativa. However, the geographic origin and the domestication times of O. sativa in China are still under debate. To settle these disputes, six chloroplast loci and four mitochondrial loci were selected to examine the relationships between 50 accessions of Asian cultivated rice and 119 accessions of common wild rice from China based on DNA sequence analysis in the present study. The results indicated that Southern China is the genetic diversity center of O. rufipogon and it might be the primary domestication region of O. sativa. Molecular dating suggested that the two subspecies had diverged 0.1 million years ago, much earlier than the beginning of rice domestication. Genetic differentiations and phylogeography analyses indicated that indica was domesticated from tropical O. rufipogon while japonica was domesticated from O. rufipogon which located in higher latitude. These results provided molecular evidences for the hypotheses of (i) Southern China is the origin center of O. sativa in China and (ii) the two subspecies of O. sativa were domesticated multiple times.

Emodin, a major active anthraquinone, frequently interacts with other drugs. As changes of efflux transporters on intestine are one of the essential reasons why the drugs interact with each other, a validated Ussing chamber technique was established to detect the interactions between emodin and efflux transporters, including P-glycoprotein (P-gp), multidrug-resistant associated protein 2 (MRP2), and multidrug-resistant associated protein 3 (MRP3). Digoxin, pravastatin, and teniposide were selected as the test substrates of P-gp, MRP2, and MRP3. Verapamil, MK571, and benzbromarone were their special inhibitors. The results showed that verapamil, MK571, and benzbromarone could increase digoxin, pravastatin, and teniposide absorption, and decrease their Er values, respectively. Verapamil (220 μM) could significantly increase emodin absorption at 9.25 μM. In the presence of MK571 (186 μM), the Papp values of emodin from M-S were significantly increased and the efflux ratio decreased. With the treatment of emodin (185, 370, and 740 μM), digoxin absorption was significantly decreased while teniposide increased. These results indicated that emodin might be the substrate of P-gp and MRP2. Besides, it might be a P-gp inducer and MRP3 inhibitor on enterocyte, which are reported for the first time. These results will be helpful to explain the drug-drug interaction mechanisms between emodin and other drugs and provide basic data for clinical combination therapy.

Porcine reproductive and respiratory syndrome virus (PRRSV) 1 and PRRSV 2 have coexisted in China for a very long time. In this study, the complete genomic characterization of a PRRSV 1 strain named KZ2018 was conducted. The results showed that it shared 88.6% identity with Lelystad virus and 81.9–90.8% identities with other Chinese PRRSV 1 strains. Further study showed that its nsp2 protein had a unique discontinuous 6-amino acid (aa) deletion (aa357-360+aa411+aa449). Additionally, its GP3 and GP4 contained a long continuous 18-aa deletion in their overlapped region, which has never been described in other Chinese PRRSV 1 isolates. Amino acid analysis of cell epitopes revealed that GP3245-256 and GP457-68 were the most variable epitopes among different Chinese PRRSV 1 isolates. The results might enrich our knowledge of PRRSV 1 strains in China.

Although morphology and grain size are important to rice growth and yield, the identity of abundant natural allelic variations that determine agronomically important differences in crops is unknown. Here, we characterized the function of mitogen-activated protein kinase 3 from Oryza officinalis Wall. ex Watt encoded by OrMKK3. Different alternative splicing variants occurred in OrMKK3. Green fluorescent protein (GFP)-OrMKK3 fusion proteins localized to the cell membrane and nuclei of rice protoplasts. Overexpression of OrMKK3 influenced the expression levels of the grain size-related genes SMG1, GW8, GL3, GW2, and DEP3. Phylogenetic analysis showed that OrMKK3 is well conserved in plants while showing large amounts of variation between indica, japonica, and wild rice. In addition, OrMKK3 slightly influenced brassinosteroid (BR) responses and the expression levels of BR-related genes. Our findings thus identify a new gene, OrMKK3, influencing morphology and grain size and that represents a possible link between mitogen-activated protein kinase and BR response pathways in grain growth.The online version contains supplementary material available at 10.1007/s12374-020-09290-2.

The brown planthopper (BPH) is a serious insect pest of rice and a substantial threat to rice production. Identification of new BPH resistance genes and their transfer into modern rice cultivars are effective breeding approaches to reduce the damage caused by BPH. In this study, we mapped a BPH resistance gene to a 50-kb genomic interval between two InDel markers 4M03980 and 4M04041 on the short arm of chromosome 4 in indica rice cultivar BP60, where the BPH resistance gene was mapped in Rathu Heenati by Liu et al. (2015) and named “Bph3”. This region contains two annotated genes Os04g0201900 and Os04g0202300, which encode lectin receptor kinases responsible for BPH resistance. We also developed a molecular marker “MM28T” for Bph3, and introgression Bph3 into susceptible rice restorer lines Guihui582 and Gui7571 by the marker-assisted selection (MAS) approach. The BPH resistance level is significantly enhanced in the Bph3-introgression lines, the resistance scores decrease from 8.2 to 3.6 for Guihui582 and decrease from 8.7 to around 3.8 for Gui7571. Therefore, developing molecular markers for the BPH resistance gene Bph3 and using them for molecular breeding will facilitate the creation of BPH-resistance rice cultivars to reduce damage caused by BPH.

Abstract The Chinese drug pair Danshen ( Salvia miltiorrhiza )–Sanqi ( Panax ginseng ) has been widely used for centuries treating various cardiovascular disorders, among which salvianlic acid B (SAB), ginsenoside Rg 1 (GRg 1 ), ginsenoside Rb 1 (GRb 1 ) and notoginsenoside R 1 (NGR 1 ) were identified as the major components. The present study focused on the interaction between these components based on investigating their intestinal absorption using the Ussing chamber technique. The concentrations of SAB, GRg 1 , GRb1 and NGR 1 in the intestinal perfusate were determined by LC–MS/MS method, followed by Q (accumulative quantity) and P app (apparent permeability). The results showed that all these four main components displayed very low permeabilities, which implied their poor absorption in the rat intestine. The intestinal absorption level of SAB displayed regioselectivity: duodenum &lt; jejunum &lt; ileum. However, there was no significant difference in the absorption of GRg 1 and GRb 1 in the different segments. The Q and P app values of the four main components were obviously increased in jejunum when co‐administrating Danshen extract with Sanqi extract. In conclusion, compatibility of Danshen and Sanqi could remarkably improve the intestinal absorption level of the main components in the pair. To some extent, this might explain the nature of the compatibility mechanisms of composite formulae in TCMs.

The root of Reynoutria multiflora (Thunb.) Moldenke (syn.: Polygonum multiflorum Thunb., HSW) is a distinguished herb that has been popularly used in traditional Chinese medicine (TCM). Evidence of its potential side effect on liver injury has accumulated and received much attention. The objective of this study was to profile the metabolic characteristics of lipids in injured liver of rats induced by HSW and to find out potential lipid biomarkers of toxic consequence. A lipopolysaccharide (LPS)-induced rat model of idiosyncratic drug-induced liver injury (IDILI) was constructed and evident liver injury caused by HSW was confirmed based on the combination of biochemical, morphological, and functional tests. A lipidomics method was developed for the first time to investigate the alteration of lipid metabolism in HSW-induced IDILI rat liver by using ultra-high-performance liquid chromatography/Q-exactive Orbitrap mass spectrometry coupled with multivariate analysis. A total of 202 characterized lipids, including phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylethanolamine (PE), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), phosphatidylinositol (PI), lysophosphatidylinositol (LPI), phosphatidylserine (PS), phosphoglycerols (PG), and ceramide (Cer), were compared among groups of LPS and LPS + HSW. A total of 14 out 26 LPC, 22 out of 47 PC, 19 out of 29 LPE, 16 out of 36 PE, and 10 out of 15 PI species were increased in HSW-treated rat liver, which indicated that HSW may cause liver damage via interfering the phospholipid metabolism. The present work may assist lipid biomarker development of HSW-induced DILI and it also provide new insights into the relationships between phospholipid perturbation and herbal-induced idiosyncratic DILI.

Selective separation of chemical components from seven kinds of volatile oil by countercurrent chromatography with three types of cyclodextrins as selective reagent was investigated in this work. Preparative separation of chemical components from volatile oil is generally quite challenging due to their extremely complexity of the composition. A biphasic solvent system n-hexane-0.10 mol L(-1) cyclodextrin (1:1, v/v) was selected for separation of components from volatile oil and three types of cyclodextrins were investigated, including β-cyclodexrin, methyl-β-cyclodexrin and hydroxypropyl-β-cyclodexrin. All kinds of volatile oils are from seven kinds of traditional Chinese herb. Results showed that some chemical components could be well separated with high purity from each kind of volatile oil using different type of cyclodextrin as selective reagent. For example, germacrone and curcumenone could be selectively separated from volatile oil of Curcumae Rhizoma with methyl-β-cyclodexrin and hydroxypropyl-β-cyclodexrin as selector respectively, and other five components were selectively separated from volatile oil of Chuanxiong Rhizoma, Myristicae Semen, Aucklandiae Radix and Angelicae Sinensis Radix by countercurrent chromatography with different cyclodexrin as selective reagent. Separation mechanism for separation of components from volatile oil by countercurrent chromatography with cyclodextrin as selective reagent was proposed. Peak resolution of the present separation method could be greatly influenced by the chemical compositions of volatile oil.

Abstract Over the past 30 years, human disturbance and habitat fragmentation have severely endangered the survival of common wild rice ( Oryza rufipogon Griff.) in China. A better understanding of the genetic structure of O. rufipogon populations will therefore be useful for the development of conservation strategies. We examined the diversity and genetic structure of natural O. rufipogon populations at the national, provincial, and local levels using simple sequence repeat (SSR) markers. Twenty representative populations from sites across China showed high levels of genetic variability, and approximately 44% of the total genetic variation was among populations. At the local level, we studied fourteen populations in Guangxi Province and four populations in Jiangxi Province. Populations from similar ecosystems showed less genetic differentiation, and local environmental conditions rather than geographic distance appeared to have influenced gene flow during population genetic evolution. We identified a triangular area, including northern Hainan, southern Guangdong, and southwestern Guangxi, as the genetic diversity center of O. rufipogon in China, and we proposed that this area should be given priority during the development of ex situ and in situ conservation strategies. Populations from less common ecosystem types should also be given priority for in situ conservation.

Rapeseed has the ability to absorb cadmium in the roots and transfer it to aboveground organs, making it a potential species for remediating soil cadmium (Cd) pollution. However, the genetic and molecular mechanisms underlying this phenomenon in rapeseed are still unclear. In this study, a 'cadmium-enriched' parent, 'P1', with high cadmium transport and accumulation in the shoot (cadmium root: shoot transfer ratio of 153.75%), and a low-cadmium-accumulation parent, 'P2', (with a cadmium transfer ratio of 48.72%) were assessed for Cd concentration using inductively coupled plasma mass spectrometry (ICP-MS). An F2 genetic population was constructed by crossing 'P1' with 'P2' to map QTL intervals and underlying genes associated with cadmium enrichment. Fifty extremely cadmium-enriched F2 individuals and fifty extremely low-accumulation F2 individuals were selected based on cadmium content and cadmium transfer ratio and used for bulk segregant analysis (BSA) in combination with whole genome resequencing. This generated a total of 3,660,999 SNPs and 787,034 InDels between these two segregated phenotypic groups. Based on the delta SNP index (the difference in SNP frequency between the two bulked pools), nine candidate Quantitative trait loci (QTLs) from five chromosomes were identified, and four intervals were validated. RNA sequencing of 'P1' and 'P2' in response to cadmium was also performed and identified 3502 differentially expressed genes (DEGs) between 'P1' and 'P2' under Cd treatment. Finally, 32 candidate DEGs were identified within 9 significant mapping intervals, including genes encoding a glutathione S-transferase (GST), a molecular chaperone (DnaJ), and a phosphoglycerate kinase (PGK), among others. These genes are strong candidates for playing an active role in helping rapeseed cope with cadmium stress. Therefore, this study not only sheds new light on the molecular mechanisms of Cd accumulation in rapeseed but could also be useful for rapeseed breeding programs targeting this trait.

Grain size is one of the most important agronomic traits for grain yield determination in rice. To better understand the proteins that are regulated by the grain size regulatory gene OsMKK3, this gene was knocked out using the CRISPR/Cas9 system, and tandem mass tag (TMT) labeling combined with liquid chromatograph-tandem mass spectrometry analysis was performed to study the regulation of proteins in the panicle. Quantitative proteomic screening revealed a total of 106 differentially expressed proteins (DEPs) via comparison of the OsMKK3 mutant line to the wild-type YexiangB, including 15 and 91 up-regulated and down-regulated DEPs, respectively. Pathway analysis revealed that DEPs were enriched in metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and photosynthesis. Strong interactions were detected among seven down-regulated proteins related to photosystem components in the protein-protein interaction network, and photosynthetic rate was decreased in mutant plants. The results of the liquid chromatography-parallel reaction monitoring/mass spectromery analysis and western blot analysis were consistent with the results of the proteomic analysis, and the results of the quantitative reverse transcription polymerase chain reaction analysis revealed that the expression levels of most candidate genes were consistent with protein levels. Overall, OsMKK3 controls grain size by regulating the protein content in cells. Our findings provide new candidate genes that will aid the study of grain size regulatory mechanisms associated with the mitogen-activated protein kinase (MAPK) signaling pathway.

Prior studies indicate that subjects undergoing methadone maintenance therapy (MMT) may experience anxiety, depression and cravings. This study aimed to explore the impact of intermittent theta burst stimulation (iTBS)-MMT combination on craving and emotional symptoms of opioid use disorder. This comparative study included subjects with opioid use disorder at the Methadone Maintenance Clinic of Pudong New Area between September 2019 and March 2020. The subjects were divided into two groups: those who received iTBS-MMT combination treatment (iTBS-MMT) and those who received MMT treatment and sham stimulation treatment (MMT). Outcomes were reduction rate of anxiety, depression and craving. Anxiety was measured by Hamilton Anxiety (HAMA) scale, depression was determined by Hamilton Depression (HAMD) scale and craving was analyzed using visual analog scale. A total of 76 subjects completed the treatment, with 38 subjects in each group. Twenty days after treatment, subjects in the iTBS-MMT group had significant improvement of anxiety (HAMA reduction rate), depression (HAMD reduction rate) and craving (Craving reduction rate) reduction rate compared with MMT group. iTBS-MMT combination treatment may produce better drug craving reduction and emotional improvement than MMT alone.

Herb genomics and comparative genomics provide a global platform to explore the genetics and biology of herbs at the genome level. Panax ginseng C.A. Meyer is an important medicinal plant for a variety of bioactive chemical compounds of which the biosynthesis may involve transport of a wide range of substrates mediated by oligopeptide transporters (OPT). However, information about the OPT family in the plant kingdom is still limited. Only 17 and 18 OPT genes have been characterized for Oryza sativa and Arabidopsis thaliana, respectively. Additionally, few comprehensive studies incorporating the phylogeny, gene structure, paralogs evolution, expression profiling, and co-expression network between transcription factors and OPT genes have been reported for ginseng and other species. In the present study, we performed those analyses comprehensively with both online tools and standalone tools. As a result, we identified a total of 268 non-redundant OPT genes from 12 flowering plants of which 37 were from ginseng. These OPT genes were clustered into two distinct clades in which clade-specific motif compositions were considerably conservative. The distribution of OPT paralogs was indicative of segmental duplication and subsequent structural variation. Expression patterns based on two sources of RNA-Sequence datasets suggested that some OPT genes were expressed in both an organ-specific and tissue-specific manner and might be involved in the functional development of plants. Further co-expression analysis of OPT genes and transcription factors indicated 141 positive and 11 negative links, which shows potent regulators for OPT genes. Overall, the data obtained from our study contribute to a better understanding of the complexity of the OPT gene family in ginseng and other flowering plants. This genetic resource will help improve the interpretation on mechanisms of metabolism transportation and signal transduction during plant development for Panax ginseng.

Low temperature is one of the important environmental factors that affect rice growth and yield. To better understand the japonica rice responses to cold stress, isobaric tags for a relative and absolute quantification (iTRAQ) labeling-based quantitative proteomics approach was used to detected changes in protein levels. Two-week-old seedlings of the cold tolerant rice variety Kongyu131 were treated at 8°C for 24, 48 and 72 h, then the total proteins were extracted from tissues and used for quantitative proteomics analysis. A total of 5082 proteins were detected for quantitative analysis, of which 289 proteins were significantly regulated, consisting of 169 uniquely up-regulated proteins and 125 uniquely down-regulated proteins in cold stress groups relative to the control group. Functional analysis revealed that most of the regulated proteins are involved in photosynthesis, metabolic pathway, biosynthesis of secondary metabolites and carbon metabolism. Western blot analysis showed that protein regulation was consistent with the iTRAQ data. The corresponding genes of 25 regulated proteins were used for quantitative real time PCR analysis, and the results showed that the mRNA level was not always parallel to the corresponding protein level. The importance of our study is that it provides new insights into cold stress responses in rice with respect to proteomics and provides candidate genes for cold-tolerance rice breeding.

Porcine reproductive and respiratory syndrome virus has caused hundreds of thousands of deaths in pig farms in many swine-producing areas in the world in recent years. However, at present there is no effective method to prevent and control the disease, and there is a need to develop new antiviral strategies. In this study, four recombinant adenoviruses expressing shRNAs targeting ORF1b, ORF5 and ORF7 were constructed, and it was found that they could down-regulate effectively specific gene expression and inhibit viral replication in MARC-145 cells when compared to the controls. They could also inhibit effectively PRRSV replication in porcine alveolar macrophages. The inhibition effect was dose-dependent and could be sustained for at least 96h in macrophages. In addition, PRRSV replication could be suppressed significantly by shRNA in cells infected previously or simultaneously with PRRSV. The results indicated that the shRNA-expressing rAd5 targeting to various gene regions of PRRSV might be a potential anti-PRRSV strategy.

Abstract The root of Polygonum multiflorum (PM) is an important Chinese herbal medicine for treatment of various diseases. Extensive pharmacological studies have been conducted and demonstrated that it shows a wide range of bioactivities. Meanwhile, a considerable number of hepatotoxicity cases owing to oral administration of PM have been reported and have attracted great attention. However, the limited knowledge regarding the metabolism of PM restricts the deeper studies on its pharmacological/toxicological mechanism and therapeutic material basis. The present study aimed to develop a high‐performance liquid chromatography coupled with a linear ion trap–Orbitrap hybrid mass spectrometry method for separation and identification of metabolites in rat urine and plasma after oral administration of PM . Based on the proposed strategy, metabolism profiles of PM in rats were proposed for the first time and 43 metabolites were characterized or tentatively identified. Phase II metabolism, such as glucuronidation and sulfation, are the principal pathways of the main components. These findings will be beneficial to further understanding of the pharmacological mechanism and pharmacodynamic material basis of PM.

The new crown pneumonia (COVID-19) epidemic in 2020 has spread globally, causing schools around the world to stop routine teaching. Educational institutions in various countries have adopted online teaching methods in response to this crisis. This research, carried out in Human Institute of Information Technology with a number of teachers and students as its subjects, sets out to give statistical analysis upon the students' selections of online teaching platforms as well as their evaluations of online teaching. At the same time, based on the online teaching practice of “Building Structure”, a certain quantity of research upon the online teaching practice is completed among the students who began their college studies in engineering cost in the year of 2018. According to all these studies, it is evident that multiple factors such as teachers' ages, professions, and the features of various online teaching platforms, can determine which one is used by different individuals. The evaluation results suggest that online teaching is necessary under the impact of the epidemic despite the fact that students may face a series of problems for lack of self-control and other possible reasons. Through practice, an innovative teaching and evaluation method can partially solve the problems found in online teaching and provide useful ideas for creating higher quality teaching on the Internet.

Amomum villosum Lour. is the plant that produces the famous traditional Chinese medicine Amomi Fructus. Frequent habitat destruction seriously threatens A. villosum germplasm resources. Genetic diversity is very important to the optimization of germplasm resources and population protection, but the range of inherited traits within A. villosum is unclear. In this study, we analyzed the genetic diversity and genetic structures of A. villosum populations in Guangdong and constructed a local reference DNA barcode library as a resource for conservation efforts.DNA barcoding and Inter-Simple Sequence Repeat (ISSR) markers were used to investigate the population genetics of A. villosum. Five universal DNA barcodes were amplified and used in the construction of a DNA barcode reference library. Parameters including percentage of polymorphic sites (PPB), number of alleles (Na), effective number of alleles (Ne), Nei's gene diversity index (H), and Shannon's polymorphism information index (I) were calculated for the assessment of genetic diversity. Genetic structure was revealed by measuring Nei's gene differentiation coefficient (Gst), total population genetic diversity (Ht), intra-group genetic diversity (Hs), and gene flow (Nm). Analysis of molecular variance (AMOVA), Mantel tests, unweighted pair-group method with arithmetic mean (UPGMA) dendrogram, and principal co-ordinates (PCoA) analysis were used to elucidate the genetic differentiation and relationship among populations.A total of 531 sequences were obtained from the five DNA barcodes with no variable sites from any of the barcode sequences. A total of 66 ISSR bands were generated from A. villosum populations using the selected six ISSR primers; 56 bands, 84.85% for all the seven A. villosum populations were polymorphic. The A. villosum populations showed high genetic diversity (H = 0.3281, I = 0.4895), whereas the gene flow was weak (Nm = 0.6143). Gst (0.4487) and AMOVA analysis indicated that there is obvious genetic differentiation amongA. villosum populations and more genetic variations existed within each population. The genetic relationship of each population was relatively close as the genetic distances were between 0.0844 and 0.3347.

Black soybeans are rich in isoflavones, which have several beneficial health effects. In this study, a validated method based on UHPLC-MS/MS was developed to screen black-soybean metabolites in rat urine, bile, and plasma and to quantify the compounds (daidzein, genistein, glycitein, and daidzin) and their metabolites (daidzein-4′-β-d-glucuronide, genistein-7-β-d-glucuronide, and genistein-4′-β-d-glucuronide) in plasma. Thirty-seven compounds were tentatively detected in the biological samples. The method was fully validated in quantitative experiments, including in assessments of linearity (2.5–100 ng/mL for daidzein, genistein, and glycitein; 10–100 ng/mL for daidzin; 5–3125 ng/mL for genistein-7-β-d-glucuronide; and 5–1562.5 ng/mL for daidzein-4′-β-d-glucuronide and genistein-4′-β-d-glucuronide), matrix effects (85–115%), recovery (80–105%), precision (<10%), and accuracy (<10%). The compounds were stable throughout sample storage, treatment, and analysis. The method was first applied to detect IFs and metabolites in rats after oral administration of black-soybean extract. These results support the potential of this method for successful application in pharmacokinetic studies.

Dendrobine is a major alkaloid present mainly in dendrobium nobile Lindl. It has been reported to have analgesic, antipyretic, lower heart rate and blood pressure and other pharmacologic activities. Despite its critical pharmacological function, its metabolite profiling is still unclear.In this study, the in vivo metabolite profiling of dendrobine in rats was investigated using ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS). The metabolites were predicted using MetabolitePilotTM software with a mass defect filter (MDF) technique. These predicted metabolites were further analyzed by MS2 spectra and compared with the detailed fragmentation pathway of the dendrobine standard and literature data.Total of 59 metabolites were identified for the first time in rat plasma and urine after oral administration of dendrobine. Demethylated, dehydrogenated, hydroxylated, ketonizated and glucuronide were the major metabolic pathways.This research provides scientific and reliable support for full understanding of the metabolic fate of dendrobine in vivo.

The quantification method for the determination of kojic acid in foods using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed. For solid samples, the kojic acid was extracted with acetonitrile; for liquid samples, they were diluted with water, then deproteinized by the deposition with zinc acetate and potassium ferrocyanide. The analytes were determined by HPLC-MS/MS on a C18 column with 5 mmol/L ammonium acetate/formic acid solution as mobile phases. The analysis of kojic acid was performed under selected reaction monitoring (SRM) mode by selecting one parent ion and two daughter ions as qualitative ions with [13C6]-kojic acid as the internal standard, and the most abundant daughter ion as quantitative ion. The limits of quantification (S/N > 10) were 0.1 mg/kg for the solid samples, and 2.5 mg/kg for the liquid samples. The good linearity (r > 0.99) was achieved for the target compound over the range of 0.1 - 2.0 mg/L. The recoveries at three levels for kojic acid were from 72.6% to 114% with the relative standard deviations no more than 11.4%. The method is simple and practical, and can be applied to most of matrices which may contain kojic acid as food additives. It can meet the qualitative and quantitative requirements for import and export foods.

Isolation of viruses using chick embryos is a classical virological method. Inoculation of the allantoic cavity and use of allantoic fluid is a common method of passaging isolated avian influenza viruses. In the present study, 2490 fresh fecal samples and 4967 old fecal samples were investigated and subjected to conventional passaging (allantoic fluid method). Two newly developed methods-the allantochorion and allantoic fluid mixed method and the chick embryo and allantoic fluid mixed method-were also examined. The rates of influenza virus isolation for these three methods were compared. There appeared to be little difference among these methods when fresh fecal samples were used. However, for the old fecal samples, isolation rates for influenza virus were significantly higher for the chick embryo and allantoic fluid mixed method compared with the conventional allantoic fluid method. All viruses isolated using the conventional allantoic fluid method were isolated successfully using the two newly developed methods. These results suggest that using chick embryos in conjunction with allantoic fluid is effective for early virus isolation, especially for fecal samples that are not fresh. Additionally, practical chick embryo passage methods are described that improve significantly the rate of isolation of influenza viruses from fecal samples of migratory birds in a complex wild ecological environment.

To investigate the intestinal absorption characteristics of gastrodigenin.In vitro everted gut sac model and in situ rat single-pass intestinal perfusion model were used to evaluate the absorption characteristics of gastrodigenin in the different intestinal segments. The concentrations of gastrodigenin in the samples were determined by Ultra Performance Liquid Chromatography (UPLC) method, and the relevant absorption parameters were calculated.In the everted gut sac tests, no significant difference of absorption among the four segments was observed. A positive correlation was found between drug concentration and the accumulated absorption amount (Q). At the concentration of 400 mg x L(-1), the Q of gastrodigenin in the duodenum, jejunum, ileum and colon were 224.33, 225.81, 233.18 and 189.25 microg, respectively. The in situ rat single-pass intestinal perfusion tests showed that there was also no significant difference of absorption among the four segments. The absorption rates (A) of gastrodigenin in the duodenum, jejunum, ileum and colon were 45.8%, 48.39%, 47.00%, 54.35%, respectively.Gastrodigenin can be well absorbed via passive diffusion in the intestine. The absorption rates of gastrodigenin in the different intestinal segments show no regioselectivity.

Persistent tapetal cell1 (PTC1) plays a curial role in pollen development, and is thought to function as a transcriptional activator in rice. However, the molecular mechanism of PTC1 in regulating pollen development and its cis-elements are not well understood. We identified a novel weak male sterility mutant (ms92) which exhibited expanded tapetum and shrink pollen grains. Map-based cloning and allelic analysis suggested that the male sterility of ms92 was caused by a DNA fragment substitution in the promoter of PTC1. The decreased expression of MS92/PTC1 in ms92 and cis-element analysis indicated that the substituted sequence contained several potential binding cis-element of negative feedback. MS92/PTC1 was specifically expressed in tapetum and microspores at the young microspore stage, and its protein was localized in nucleus. We further found that MS92/PTC1 functions as a transcription activator by recognizing H3K4me3. Transcriptomic analysis revealed that a number of genes involved in tapetum degeneration and pollen wall formation were down-regulated in ms92, which are the potential targets of MS92/PTC1. The substitution fragment in MS92/PTC1 promoter was essential for pollen development, and we provided a novel mutant for further identifying the cis-elements in promoter and the molecular network of MS92/PTC1.

The root of Reynoutria multiflora (Thunb.) Moldenke (syn: Polygonum multiflorum Thunb.) is a distinguished herb that has been popularly used in traditional Chinese medicine. The raw Reynoutria multiflora (RRM) should be processed by steaming before use, and the processing time is not specified in the processing specification. Our previous studies showed that the efficacy and toxicity of processed Reynoutria multiflora (PRM) at different processing times were inconsistent. A comprehensive identification method was established in this study to find a quality marker of raw Reynoutria multiflora (RRM) and processed Reynoutria multiflora (PRM) with different processing times. Metabolomics based on ultra-high-performance liquid chromatography tandem quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive plus orbitrap MS/MS) was used in this study. Using the CD.2 software processed database, multivariate statistical analysis methods coupled with cluster analysis and heatmap were implemented to distinguish between RRMs and PRMs with different processing times. The results showed that RRM and PRMs processed for 4, 8, 12, and 18 h cluster into group 1, and PRM processed for 24 and 32 h into group 2, indicating that it can effectively distinguish between the two groups and twenty potential markers, made the highest contributions to the observed chemical differences between two groups. Among them, tetrahydroxystilbene-O-hexoside-O-galloyl and sucrose can be used to identify PRM processed for 24 h. Therefore, the properties of RRM changed after 24 h of processing, and the quality markers were screened to distinguish RRM and PPM. It can also be used as an important control technology for the processing of RM, which has wide application prospects.

The root of Reynoutria multiflora Thunb. Moldenke (RM, syn.: Polygonum multiflorum Thunb.) has been widely used in TCM clinical practice for centuries. The raw R. multiflora (RRM) should be processed before use, in order to reduce toxicity and increase efficiency. However, the content of trans-2, 3, 5, 4'-tetrahydroxystilbene-2-O-β-D-glucopyranoside (trans-THSG), which is considered to be the main medicinal ingredient, decreases in this process. In order to understand the changes of stilbene glycosides raw R. multiflora (RRM) and processed R. multiflora (PRM), a simple and effective method was developed by ultra high performance liquid chromatography tandem quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive plus orbitrap MS/MS). The content and quantity of stilbene glycosideshave undergone tremendous changes during the process. Seven parent nucleus of stilbene glycosides and 55 substituents, including 5-HMF and a series of derivatives, were identified in PM. 146 stilbene glycosides were detected in RRM, The number of detected compounds increased from 198 to 219 as the processing time increased from 4 to 32 h. Among the detected compounds, 102 stilbene glycosides may be potential new compounds. And the changing trend of the compounds can be summarized in 3 forms: gradually increased, gradually decreased, first increased and then decreased or decreased first. The content of trans-THSG was indeed decreased during processing, as it was converted into a series of derivatives through the esterification reaction with small molecular compounds. The clarification of secondary metabolite group can provide a basis for the follow-up study on the mechanism of pharmacodynamics and toxicity of PM, and for screening of relevant quality markers.

Ginsenosides have poor oral bioavailability and undergo rapid biological transformation in the complex gastrointestinal environment. Most studies on the metabolism of ginsenosides have focused on gut bacteria, yet gastric juice remains a nonnegligible factor. Metabolic profiles of ginsenoside monomers formed in artificial gastric juice were separately investigated and qualitatively identified using ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MSn ). A common pattern of their metabolic pathways was established, showing that ginsenosides were transformed via deglycosylation, hydration, and dehydration pathways. Two major structure types, 20(S), 20(R)-protopanaxatriols and 20(S), 20(R)-protopanaxadiols, basically shared similar transformation pathways and yielded deglycosylated, hydrated, and dehydrated products. Fragmentation patterns of major ginsenosides were also discussed. Consequently, gastric juice, as the primary link in ginsenoside metabolism and as important as the intestinal flora, produces considerable amounts of degraded ginsenosides, providing a partial explanation for the low bioavailabilities of primary ginsenosides.

The content changes of 14 components of the Huangqi–Sanqi herb pair in the decoction process were compared and evaluated.

Canine distemper (CD) is a highly contagious viral disease worldwide. Although live attenuated vaccine is available as a preventive measure against the disease, cases of vaccination failure highlight the importance of potential alternative agent against canine distemper virus (CDV). CDV infects cells mainly by binding signaling lymphocyte activation molecule (SLAM) and Nectin-4 receptor. Here, to develop a new and safe antiviral biological agent for CD, we constructed and expressed CDV receptor proteins fused with Fc region of canine IgG-B, namely, SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc in HEK293T cells, and antiviral activity of these receptor-Fc proteins was subsequently evaluated. The results showed that the receptor-Fc proteins efficiently bound to receptor binding domain (RBD) of CDV-H, meanwhile, these receptor-Fc proteins competitively inhibited the binding of His-tagged receptor proteins (SLAM-His or Nectin-His) to CDV-H-RBD-Flag protein. Importantly, receptor-Fc proteins exhibited potent anti-CDV activity in vitro. Treatment with receptor-Fc proteins at the pre-entry stage dramatically suppressed CDV infectivity in Vero cells stably expressing canine SLAM. The minimum effective concentration (MEC) of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was 0.2 μg/mL, 0.2 μg/mL, 0.02 μg/mL. The 50% inhibition concentration (IC50) of three proteins was 0.58 μg/mL, 0.32 μg/mL and 0.18 μg/mL, respectively. Moreover, treatment with receptor-Fc proteins post viral infection can also inhibit CDV reproduction, the MEC of SLAM-Fc, Nectin-Fc and SLAM-Nectin-Fc was same as pre-treatment, and the IC50 of receptor-Fc proteins was 1.10 μg/mL, 0.99 μg/mL and 0.32 μg/mL, respectively. The results suggested that the receptor-Fc proteins were more effective for pre-entry treatment than post-infection treatment, furthermore, SLAM-Nectin-Fc was more effective than SLAM-Fc and Nectin-Fc. These findings revealed the receptor-Fc proteins were promising candidates as inhibitor against CDV.

Generally, chloroplast genomes of angiosperms are always highly conserved but carry a certain number of variation among species. In this study, chloroplast genomes of 13 species from Datureae tribe that are of importance both in ornamental gardening and medicinal usage were studied. In addition, seven chloroplast genomes from Datureae together with two from Solanaceae species retrieved from the National Center for Biotechnology Information (NCBI) were integrated into this study. The chloroplast genomes ranged in size from 154,686 to 155,979 and from 155,497 to 155,919 bp for species of Datura and Brugmansia , respectively. As to Datura and Brugmansia , a total of 128 and 132 genes were identified, in which 83 and 87 protein coding genes were identified, respectively; Furthermore, 37 tRNA genes and 8 rRNA genes were both identified in Datura and Brugmansia. Repeats analysis indicated that the number and type varied among species for Simple sequence repeat (SSR), long repeats, and tandem repeats ranged in number from 53 to 59, 98 to 99, and 22 to 30, respectively. Phylogenetic analysis based on the plastid genomes supported the monophyletic relationship among Datura and Brugmansia and Trompettia , and a refined phylogenic relationships among each individual was resolved. In addition, a species-specific marker was designed based on variation spot that resulted from a comparative analysis of chloroplast genomes and verified as effective maker for identification of D. stramonium and D. stramonium var. inermis . Interestingly, we found that 31 genes were likely to be under positive selection, including genes encoding ATP protein subunits, photosystem protein subunit, ribosome protein subunits, NAD(P)H dehydrogenase complex subunits, and clp P, pet B, rbc L, rpo Cl, ycf 4, and cem A genes. These genes may function as key roles in the adaption to diverse environment during evolution. The diversification of Datureae members was dated back to the late Oligocene periods. These chloroplast genomes are useful genetic resources for taxonomy, phylogeny, and evolution for Datureae.

Additional file 1: Table s1. Information on the Chinese reference strains used in this study

A method was established for the determination of deoxynivalenol (vomitoxin) in grain and its products based on solid-phase extraction coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was firstly extracted by acetonitrile-water (84:16, v/v). The extract was then cleaned-up by an HLB solid phase extraction cartridge. The separation was carried out on a Phenomenex Kinetex C18 column (100 mm x4. 6 mm, 2.6 microm) with a gradient elution using 0.3% per hundred ammonia solution-acetonitrile as mobile phases. The analysis of deoxynivalenol was performed under electrospray negative ionization mode. The limit of detection (LOD, S/N= 3) and the limit of quantification (LOQ, S/N = 10) were 20 microg/kg and 50 microg/kg, respectively. A good linearity (r > 0.99) was achieved for the target compound over the range of 20-1000 pg/L. The recoveries at the three spiked levels (50, 100, 500 microg/kg) in the blank matrices such as flour, barley, soybean, rice, cornmeal, cassava and wheat, were varied from 75.6% to 111.0% with the relative standard deviations no more than 13. 0%. The method is accurate, efficient, sensitive and practical. The cost of pretreatment is obviously reduced by replacing immunoaffinity columns and Mycosep columns with HLB columns which have the same purification effect.

2,3,5,4'-Tetrahydroxystilbene-2-O-β-d-glucoside (THSG), a polyphenol stilbene compound, is the main active constituent in Polygonum multiflorum. In this study, a comprehensive analytical strategy was developed for the characterization of THSG metabolites in vivo (rat plasma, bile, urine, heart, liver, spleen, lung, kidney, and stomach) utilizing ultrahigh performance liquid chromatography coupled with Q Exactive hybrid quadrupole-Orbitrap mass spectrometry (UHPLC-Q Exactive-Orbitrap MS) based on multiple data-processing techniques. As a result, a total of 75 metabolites were characterized in bio-samples, and calculated Clog P values were further employed to assign the chemical structures of some isomers. Glucoside hydrolysis, hydrogenation, hydroxylation, glucuronide conjugation, and sulfate conjugation would be the major metabolic pathways of THSG. It appeared that most metabolites would generally undergo phase I reactions followed by phase II reactions. These results provided valuable information for in-depth understanding of the safety and efficacy of THSG and showed a valuable methodology for metabolic characterization.

Dissection of the genetic basis of heterosis facilitates hybrid breeding in crops. In this study, heterotic loci (HL) for seven yield and yield-related traits were identified in a set of maize introgression lines (ILs) under two environments (locations)by a backcross population with one of their parents, Zong 3. Middle parent heterosis of seven yield and yield-related traits was calculated based on the field experiments conducted at two locations. A total of 120 significant loci for six traits were identified by calculating marker-trait coefficients with the software Graphical Genotypes (GGT) 2.0, and 48 loci represent for five traits were commonly identified under two environments. This indicates that it is possible to identify HL with the population derived from ILs. Of the 48 significant loci, 13were found to be associated with more than 2 different traits, indicating strong genetic correlations among these traits. For yield and most of the yield-related traits, the heterozygosity of the markers at the HL were strongly correlated to the performance of middle parent heterosis, suggesting that the HL identified in this study could be used for heterosis prediction and maker-assisted selection in maize hybrid breeding.

&lt;p&gt;&lt;em&gt;The dust-depressors have been developed utilizing a method of microbial induced carbonate precipitation. This microbial dust-depressor has the characteristics of high efficiency for dust suppression and environmental protection. Optimal compositions of urease dust-depressor and microbial dust-depressor have been studied. In addition, pure water, CaCl2 and super absorbent polymer are chosen to compare with new dust-depressors on the performances. The results showed that the microbial dust-depressor had 3.13 mm/min of seepage velocity and 79.1% of dust suppression efficiency, which were superior to other dust-depressors on the performance of seepage velocity and dust suppression efficiency.&lt;/em&gt;&lt;em&gt;&lt;/em&gt;&lt;/p&gt;

Abstract Kernel weight is an important yield component in maize that was selected during domestication. Many kernel weight genes have been identified through mutant analysis, and are mostly involved in the biogenesis and functional maintenance of organelles or other fundamental cellular activities. However, only a limited number of loci underlying quantitative variation in kernel weight have been cloned. Here we characterize a maize kernel weight QTL, qKW9 and find that it encodes a DYW motif pentatricopeptide repeat protein involved in C-to-U editing of NdhB, a subunit of the chloroplast NADH dehydrogenase-like complex. In a null qKW9 background, C-to-U editing of NdhB was abolished, and photosynthesis was reduced, suggesting that qKW9 regulates kernel weight by controling the maternal source of photosynthate for grain filling. Characterization of qKW9 highlights the importance of optimizing photosynthesis on maize grain yield production.

A method was established for the determination of streptomycin (STR) and dihydrostreptomycin (DHS) in pollens based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The sample was extracted and cleaned-up by a C18 solid phase extraction cartridge. The separation was carried out on a Protemix WCX-NP5 column (100 mm x 2.1 mm, 5 microm) with a gradient elution using 5% (v/v) formic acid, 20 mmol/L ammonium acetate and methanol as mobile phases. The analysis of streptomycin and dihydrostreptomycin was performed under electrospray positive ionization mode. The limits of detection (LOD, S/N = 3) and limits of quantification (LOQ, S/N = 10) for the both were 5 microg/kg and 10 microg/kg, respectively. Good linearities (r > 0.99) were achieved for the target compounds over the range of 10-200 microg/L. The recoveries at three spiked levels (10, 20, 50 microg/kg) in the blank matrices, such as pollen pini, corn pollen, camellia pollen, sunflower pollen, rape pollen and bee pollen, were from 76.8% to 100.3% with the relative standard deviations varied from 3.70% to 12.6%. The method is accurate, practical, and can be applied to most of the contaminated matrices. With this method, heptafluorobutyric acid is not required as mobile phase which is harmful to MS spectrometer.

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Objective:To optimize the ethanol reflux extraction technology of Herba Ephedrae.Methods:In this research,Taguchi Experimental Design was carried out to investigate the ethanol concentration、the ethanol quantity、extraction time and times、in order to optimized the extract conditions,the study used the content of ephedrine hydrochloride as markers.Results:The optimized ethanol reflux extraction technology was that the 60% ethanol quantity is 6 times as much as the weight of the drugs were added,extracted 3 times,with 1.0 hours for each time.Conclusion:The optimized extraction technology was stable and feasible.

OBJECTIVE To intestigate the extraction and inclusion condition of volatile oil from Qinghao(Artemisia apiacea) and Chuanxiong(Ligusticum wallichii) in Huanghao Xiaoyan granules.METHODS The study were carried out with orthogonal experiment.With the volume of volatile oil as the target markers,the optimum conditions were determined for extraction of the volatile oil.With the recovery rate of inclusion and the volatile oil inclusion rate as the target marker the conditions for including the oil were optimized.RESULTS The optimized extraction conditions were:the water was 10 times,the medical material should be extracted for 8 h without soaking.The optimal inclusion conditions were: applying the saturated aqueous solution method,the proportion of β-CD and oil was 6∶1,temperature was 50 ℃ for inclusion,and the time was 90 min.CONCLUSION The optimum conditions for extra-ction and inclusion are steady and feasible.

OBJECTIVE To investigate the absorption characteristics of febuxostat,a new antipodagrics in rat intestine,so as to provide scientific basis for the formulation design of oral preparation of febuxostat.METHODS The absorption kinetics of febuxosatat in various intestinal segments was measured using in situ rat single-pass intestinal perfusion model and the drug concentration was analyzed by ultra performance liquid chromatography(UPLC).The effect of drug concentration on the intestinal absorption was investigated too.RESULTS The Ra,Peff and Pw values of febuxostat in duodenum and colon were significantly higher than those in jejunum and ileum(P0.05).No significant difference in the parameters was found between middle concentration(10 μmol·L-1) group and high concentration(20 μmol·L-1) group(P0.05).However,both of them were obviously higher than those in low concentration(5 μmol·L-1) group(P0.05).CONCLUSION Febuxostat can be well absorbed in the intestine.The absorption rates of febuxostat in duodenum and colon are significantly higher than those in ileum and jejunum.Efflux transport protein may be involved in the intestinal absorption of febuxostat.

A method for the determination of chloramphenicol in propolis was developed by high performance liquid chromatography-tandem mass spectrometry. The flavones were removed with lead acetate solution after the extraction of the sample with water. The extract was cleaned up by liquid-liquid extraction. Internal standard method was used for quantitative analysis. The linear range was 0.05 - 2.0 microg/L and the correlation coefficient (r2) was 0.9996. The limit of detection (LOD, S/N = 3) and limit of quantitation (LOQ, S/N = 10) were 0.1 microg/kg and 0.3 microg/kg, respectively. The recoveries ranged from 70.1% to 94.0% while the intra-day precision lower than 10% and inter-day precision lower than 15%. The method reduced the interference by removing most of the flavones and was suitable for the determination of chloramphenicol in propolis.

This paper expounded meaning of conservation of wild rice.Principal progress on in situ and ex situ conservation of wild rice in Guangxi were discussed.We bring up problems and thinking of the development in future.

The present paper is an attempt to discuss significance and primary achievements of utilizing the drought-tolerant germplasm in rice resources in Guangxi from the point of crop breeding and genetic resources.The main problems related to their exploitation in breeding and their causes have been analyzed.According to the studies,researches on drought-tolerant rice and upland rice breeding have been suggested to be paid more attention in future.In order to provide references for achieving grain security,exploitation of abundant rice resources in Guangxi is recommended,and innovations in concepts and technologies of rice breeding and drought-tolerant cultivation technologies have been discussed.

A pair of specific primers were designed according to the sequence of porcine reproductive and respiratory syndrome virus S1 ORF5 gene published in GenBank.The gene encoding PRRSV non-neutralization epitope(tGP5)was obtained by PCR.The gene was finally cloned into eukaryotic expression vector pcDNA3.1.The recombinant pcDNA3.1-tGP5 was transformed into E.coli.DH5α.The pcDNA3.1-tGP5 was succussifully constructed after identification by PCR,EcoRⅠ/HindⅢ double digestion and sequencing.

【Objective】To estimate the genetic diversity of the populations of wild rice(O.rufipogon Griff) in the whole region of Guangxi Zhuang Autonomous Region and reveal their geographically distribution characteristics.【Method】 Thirty-six SSR loci distributed on the twelve chromosomes of rice were used to study the genetic diversity of 690 wild rice individuals of 27 natural populations from 14 regions in Guangxi.【Result】 The mean number of allele(A) was 9.86 and the effective number of allele(Ae) was 5.05.The total genetic diversity(Ht) was 0.74 and the coefficient of genetic differentiation(Gst) was 0.49,which means that the populations of O.rufipogon in Guangxi has abundant genetic diversity and the genetic variability among and between the populations has the same contribution to the total diversity.The analysis of clusters showed that all the samples could be clustered into 3 groups at the coefficient of 0.5,and each group has its own geographic characteristics.【Conclusion】 O.rufipogon populations in the whole region of Guangxi was not only rich in genetic diversity,but also has close relationship with their geographical conditions like mountains,water systems and geology.

Additional file 1: Table S2.. Genbank accession numbers of DNA barcodes. Table S4. Results for one-vs-one calculation of Ka/Ks generated by KaKs_Calculator.

Additional file 8: Table S3. Genes for qRT-PCR.

The statistical characteristics of the human mobility among different cities in the spreading of COVID-19 can be described by SI spreading model. P-SI models are presented to investigate how COVID-19 spreads or diffuses in Hubei and the other 4 provinces. Based on empirical data, some experiments are then conducted under the framework of the P-SI models. The experimental results demonstrate that the P-SI models can describe the number of daily new infected people caused by COVID-19 in Hubei and the other 4 provinces according to the empirical data. In addition, the P-SI models can also predict the number of daily new infected people in the other 4 provinces after 1/24 according to the empirical data before 1/23. The P-SI models are steps toward the understanding of COVID-19’s spreading characteristics, which provides supports to isolate the source of COVID-19, to prevent and slow down the spreading of COVID-19, and to roll out effective measures for COVID-19’s prevention and control.

Abstract Background : The Amomum villosum has the situation that it is inferior and other other varieties are used as A. villosum in the market. In order to develop and utilize the genuine medicinal materials A. villosum , this experiment aims to carry out the identification and research of variety of the A. villosum and analyze its genetic diversity, constructing the DNA barcode database of the genuine medicinal materials A. villosum in Guangdong Province and providing recommendations for populations planting, which will be critical to the further research of A. villosum . (2) Methods: A total of 141 samples of A. villosum were analyzed by DNA barcoding to construct DNA barcode database. The genetic diversity of A. villosum sampled from 7 populations in Guangdong Province was detected based on ISSR molecular marker technology. (3) Results : The success rates of PCR amplification and sequencing of five barcodes of A. villosum was rbcL &gt; ITS &gt; ITS2 &gt; psbA-trnH &gt; matK . 141 samples of A. villosum from 7 populations in Guangdong Province were used to construct a reference DNA barcode database containing 531 sequences. The results of genetic diversity were as follow, the number of alleles Na ranged from 1.2879 to 1.7121, the effective number of alleles Ne ranged from 1.1848 to 1.4240, the gene diversity index (H) ranged from 0.2536 to 0.1117, and the Shannon index (I) ranged from 0.3816 to 0.1658, whichindicatedthegenetic diversity of A.Villosum was rich. The total genetic diversity among the 7 populations (Ht) was 0.3299, the genetic diversity within the populations (Hs) was 0.1819, and the gene differentiation coefficient (Gst) was 0.4487. AMOVA showed that the genetic variation within the populations and the genetic variation between the populations accounted for 68.74% (P&lt;0.05) and 31.26% (P&lt;0.05) respectively, indicating that the genetic variation of A. villosum was mainly within the populations. The gene flow Nm was 0.6143.The genetic distance of the 7 populations was 0.0844 - 0.3347, and the genetic identity was 0.7156 - 0.9191, confirming that the genetic relationship of each population was relatively close. The 7 populations were significantly grouped in the cluster analysis and the genetic level of each population from high to low was as follow: ZY (National Highway Roadside) &gt; ZJD (Zhongjiaodong) &gt; GY (Geopark) &gt;MM (Dianbai) &gt; YC (Dadong Village) &gt; XFC (Xingfu Village) &gt; TK (Tankui Village). There was no correlation between the geographic distance and the degree of genetic differentiation among populations. Conclusion : By amplifying and sequencing five barcodes of ITS2, psbA-trnH , ITS, matK and rbcL , a reference DNA barcode database of A. villosum with 531 sequences was constructed. The results of genetic diversity showed that it is necessary to take appropriate in situ protection measures for the populations of A. villosum in Yangchun City and increase the genetic exchange between populations to improve the genetic diversity of A. villosum .

Now the climate,soil and water resources situation of in situ conservation area of wild rice germplasm resources in Yulin city of Guangxi province are accorded with the ecological environment conditions of wild rice distribution site in our country,it is not polluted by and suitable for environment safety of in situ conservation for a long time.In future,we must strengthen the management of legal system,and protection works must be done well in actual.

Additional file 7: Table S2. genes were significantly differentially expressed in Y12–4.

Additional file 6: Table S1. genes were significantly differentially expressed in 253.