THP9 enhances seed protein content and nitrogen-use efficiency in maize.

Teosinte, the wild ancestor of maize (Zea mays subsp. mays), has three times the seed protein content of most modern inbreds and hybrids, but the mechanisms that are responsible for this trait are unknown1,2. Here we use trio binning to create a contiguous haplotype DNA sequence of a teosinte (Zea mays subsp. parviglumis) and, through map-based cloning, identify a major high-protein quantitative trait locus, TEOSINTE HIGH PROTEIN 9 (THP9), on chromosome 9. THP9 encodes an asparagine synthetase 4 enzyme that is highly expressed in teosinte, but not in the B73 inbred, in which a deletion in the tenth intron of THP9-B73 causes incorrect splicing of THP9-B73 transcripts. Transgenic expression of THP9-teosinte in B73 significantly increased the seed protein content. Introgression of THP9-teosinte into modern maize inbreds and hybrids greatly enhanced the accumulation of free amino acids, especially asparagine, throughout the plant, and increased seed protein content without affecting yield. THP9-teosinte seems to increase nitrogen-use efficiency, which is important for promoting a high yield under low-nitrogen conditions.

Bi-parental graph strategy to represent and analyze hybrid plant genomes.

Hybrid plants are found extensively in the wild, and they often demonstrate superior performance of complex traits over their parents and other selfing plants. This phenomenon, known as heterosis, has been extensively applied in plant breeding for decades. However, the process of decoding hybrid plant genomes has seriously lagged due to the challenges associated with genome assembly and the lack of appropriate methodologies for their subsequent representation and analysis. Here, we present the assembly and analysis of two hybrids, an intraspecific hybrid between two maize (Zea may ssp. mays) inbred lines and an interspecific hybrid between maize and its wild relative teosinte (Zea may ssp. parviglumis), utilizing a combination of PacBio High Fidelity (HiFi) sequencing and chromatin conformation capture sequencing data. The haplotypic assemblies are well-phased at chromosomal scale, successfully resolving the complex loci with extensive parental structural variations (SVs). By integrating into a bi-parental genome graph, the haplotypic assemblies can facilitate downstream short-reads-based SV calling and allele-specific gene expression analysis, demonstrating outstanding advantages over a single linear genome. Our work offers a comprehensive workflow that aims to facilitate the decoding of numerous hybrid plant genomes, particularly those with unknown or inaccessible parentage, thereby enhancing our understanding of genome evolution and heterosis.

Quantitative trait locus analysis of gray leaf spot resistance in the maize IBM Syn10 DH population.

KEY MESSAGE: The exploration and dissection of a set of QTLs and candidate genes for gray leaf spot disease resistance using two fully assembled parental genomes may help expedite maize resistance breeding. The fungal disease of maize known as gray leaf spot (GLS), caused by Cercospora zeae-maydis and Cercospora zeina, is a significant concern in China, Southern Africa, and the USA. Resistance to GLS is governed by multiple genes with an additive effect and is influenced by both genotype and environment. The most effective way to reduce the cost of production is to develop resistant hybrids. In this study, we utilized the IBM Syn 10 Doubled Haploid (IBM Syn10 DH) population to identify quantitative trait loci (QTLs) associated with resistance to gray leaf spot (GLS) in multiple locations. Analysis of seven distinct environments revealed a total of 58 QTLs, 49 of which formed 12 discrete clusters distributed across chromosomes 1, 2, 3, 4, 8 and 10. By comparing these findings with published research, we identified colocalized QTLs or GWAS loci within eleven clustering intervals. By integrating transcriptome data with genomic structural variations between parental individuals, we identified a total of 110 genes that exhibit both robust disparities in gene expression and structural alterations. Further analysis revealed 19 potential candidate genes encoding conserved resistance gene domains, including putative leucine-rich repeat receptors, NLP transcription factors, fucosyltransferases, and putative xyloglucan galactosyltransferases. Our results provide a valuable resource and linked loci for GLS marker resistance selection breeding in maize.

CYP1A2 Polymorphism and Drug Co-administration Affect the Blood Levels and Adverse Effects of Pirfenidone.

BACKGROUND: Mutations in metabolic enzymes and co-administration of drugs may affect the blood concentration of pirfenidone effective in pulmonary fibrosis. To provide a basis for the precise clinical use of pirfenidone, the authors analyzed the correlation between steady-state pirfenidone trough concentration and adverse drug reactions (ADRs) and examined the impact of CYP1A2*1C (rs2069514) and *1F (rs762551) variants and co-administration on pirfenidone blood concentrations and ADRs. METHODS: Forty-four patients were enrolled. The blood concentration of pirfenidone was determined using high-performance liquid chromatography. CYP1A2*1C and *1F genotypes were determined using direct SNP sequencing. Additional information related to drug associations was collected to screen factors affecting drug metabolism. RESULTS: The highest predictive value of ADRs was observed when the steady-state trough concentration of pirfenidone was 3.18 mcg·mL-1 and the area under the receiver operating characteristic curve was 0.701 (P = 0.024). The pirfenidone concentration-to-dose ratio (C/D) in CYP1A2*1F homozygous AA mutants was lower than that in C carriers (CC+AC) (1.28 ± 0.85 vs. 2.03 ± 1.28 mcg·mL-1; P = 0.036). Adverse drug reaction (ADR) incidence in the homozygous AA mutant group (28.0%) was significantly lower than that in the C carriers (CC+AC) (63.2%; P = 0.020), and ADR incidence in the A carriers (AC+AA) was considerably lower than that in the CC group (85.7%; P = 0.039). The C/D value of the combined lansoprazole/rabeprazole group was lower than that of the noncombination group (P < 0.05). CONCLUSIONS: The ADR incidence was positively correlated with pirfenidone blood concentration. The CYP1A2 (rs762551) AA genotype is associated with lower pirfenidone concentrations and fewer ADRs. Lansoprazole/rabeprazole co-administration reduced pirfenidone concentrations. Randomized controlled trials should further explore personalized dosing of pirfenidone and combination therapies.

The generally useful estimate of solvent systems method facilitates off-line two-dimensional countercurrent chromatography for isolating compositions from Siraitia grosvenorii roots.

Solvent system selection is a crucial and the most time-consuming step for successful countercurrent chromatography separation. A thin-layer chromatography-based generally useful estimate of solvent systems method has been developed to simplify the solvent system selection. We herein utilized the method to select a solvent system for off-line two-dimensional countercurrent chromatography to separate chemical compositions from a complex fraction of the Siraitia grosvenorii root extract. The first-dimensional countercurrent separation using chloroform/methanol/water (10:5.5:4.5, v/v/v) yielded four compounds with high purity and three mixture fractions (Fr I, III, and VII). The second-dimensional countercurrent separation conducted on Fr I, III, and VII using the hexane/ethyl acetate/methanol/water (4:6:6:4, 3:7:3:7, v/v/v) and chloroform/methanol/water (10:9:6, v/v/v) solvent systems, respectively, produced another four compounds. Four triterpenoids and four lignans were finally isolated, including two novel compounds. Hence, the generally useful estimate of solvent systems method is a feasible and efficient approach for selecting an applicable solvent system for separating complex samples. In addition, the off-line two-dimensional countercurrent chromatography method can improve both the peak resolution and the capacity of countercurrent chromatography.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon ß (IFNß), tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-ß-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

The Triterpenoids from Munronia pinnata and Their Anti-Proliferative Effects.

Six new tirucallane-type triterpenoids, named munropenes A-F (1-6), were extracted from the whole plants of Munronia pinnata using a water extraction method. Their chemical structures were determined based on detailed spectroscopic data. The relative configurations of the acyclic structures at C-17 of munropenes A-F (1-6) were established using carbon-proton spin-coupling constants (2,3JC,H) and inter-proton spin-coupling constants (3JH,H). Furthermore, the absolute configurations of munropenes A-F (1-6) were determined through high-performance liquid chromatography (HPLC), single-crystal X-ray diffraction, and electronic circular dichroism (ECD) analyses. The antiproliferative effects of munropenes A-F were evaluated in five tumor cell lines: HCT116, A549, HepG2, MCF7, and MDAMB. Munropenes A, B, D, and F (1, 2, 4, and 6) inhibited proliferation in the HCT116 cell line with IC50 values of 40.90, 19.13, 17.66, and 32.62 µM, respectively.

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography.

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane-ethyl acetate-methanol-water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol (7), sugiol (8), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one (9) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids (1, 6) and four known compounds (2-5). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.

Rhodomollosides A and B, glycosides of methyl everninate from the aerial parts of Rhododendron molle.

Two new glycosides of methyl everninate, rhodomollosides A (1) and B (2), were isolated from the aerial parts of a medicinal plant Rhododendron molle. The structures of 1 and 2 were elucidated on the basis of detailed spectroscopic analyses as well as HPLC analyses for thiazolidine derivatives of their sugar moieties. The sugar moiety of rhodomolloside A (1) was elucidated to be a rare monosaccharide, D-allose, while rhodomolloside B (2) was assigned as a D-glucoside of methyl everninate. Furthermore, they were evaluated for their cytotoxicity against RAW264.7 cells, and for their inhibitory effects with a lipopolysaccharide (LPS)-stimulated murine macrophages RAW 264.7 cells model.

Genome-wide identification and analysis of heterotic loci in three maize hybrids.

Heterosis, or hybrid vigour, is a predominant phenomenon in plant genetics, serving as the basis of crop hybrid breeding, but the causative loci and genes underlying heterosis remain unclear in many crops. Here, we present a large-scale genetic analysis using 5360 offsprings from three elite maize hybrids, which identifies 628 loci underlying 19 yield-related traits with relatively high mapping resolutions. Heterotic pattern investigations of the 628 loci show that numerous loci, mostly with complete-incomplete dominance (the major one) or overdominance effects (the secondary one) for heterozygous genotypes and nearly equal proportion of advantageous alleles from both parental lines, are the major causes of strong heterosis in these hybrids. Follow-up studies for 17 heterotic loci in an independent experiment using 2225 F2 individuals suggest most heterotic effects are roughly stable between environments with a small variation. Candidate gene analysis for one major heterotic locus (ub3) in maize implies that there may exist some common genes contributing to crop heterosis. These results provide a community resource for genetics studies in maize and new implications for heterosis in plants.

Gastric floating sustained-release tablet for dihydromyricetin: Development, characterization, and pharmacokinetics study.

Dihydromyricetin (DHM) is a natural dihydroflavonol compound with quite a number of important pharmacological properties. However, its low solubility in water and poor stability in aqueous environment, have compromised drug efficacy of DHM, thus hindering its clinical use. The present study was to develop DHM-loaded gastric floating sustained-release tablet (DHM-GFT) to improve the bioavailability of DHM. DHM-GFT was prepared via powder direct compression. The formulation of tablet was optimized in terms of the floating ability and drug release rate. The optimized DHM-GFT exhibited short floating lag time of less than 10 s and long floating duration of over 12 h in acidic medium. It had a 12-hour sustained release of DHM, which proved its potential to develop as a twice-a-day dosing preparation. The physicochemical properties of DHM-GFT well satisfied the pharmacopoeial requirements. In addition, the results from pharmacokinetic studies demonstrated that, DHM-GFT could considerably prolong the in vivo residence time of drug and improve the bioavailability via good gastric floating ability and sustained drug release when compared to DHM powder. Therefore, DHM-GFT is promising to promote the application of DHM and merits studies for further development.

Integrative analysis of DNA methylation, mRNAs, and small RNAs during maize embryo dedifferentiation.

BACKGROUND: Maize (Zea mays) is an important model crop for transgenic studies. However, genetic transformation of maize requires embryonic calli derived from immature embryo, and the impact of utilizing tissue culture methods on the maize epigenome is poorly understood. Here, we generated whole-genome MeDIP-seq data examining DNA methylation in dedifferentiated and normal immature maize embryos. RESULTS: We observed that most of the dedifferentiated embryos exhibited a methylation increase compared to normal embryos. Increased methylation at promoters was associated with down-regulated protein-coding gene expression; however, the correlation was not strong. Analysis of the callus and immature embryos indicated that the methylation increase was induced during induction of embryonic callus, suggesting phenotypic consequences may be caused by perturbations in genomic DNA methylation levels. The correlation between the 21-24nt small RNAs and DNA methylation regions were investigated but only a statistically significant correlation for 24nt small RNAs was observed. CONCLUSIONS: These data extend the significance of epigenetic changes during maize embryo callus formation, and the methylation changes might explain some of the observed embryonic callus variation in callus formation.

Genome-wide comparative analysis of digital gene expression tag profiles during maize ear development.

The present study profiled and analyzed gene expression of the maize ear at four key developmental stages. Based on genome-wide profile analysis, we detected differential mRNA of maize genes. Some of the differentially expressed genes (DEGs) were predicted to be potential candidates of maize ear development. Several well-known genes were found with reported mutant analyses, such as, compact plant2 (ct2), zea AGAMOUS homolog1 (zag1), bearded ear (bde), and silky1 (si1). MicroRNAs such as microRNA156 were predicted to target genes involved in maize ear development. Antisense transcripts were widespread throughout all the four stages, and are suspected to play important roles in maize ear development. Thus, identification and characterization of important genes and regulators at all the four developmental stages will contribute to an improved understanding of the molecular mechanisms responsible for maize ear development.

Metabolites of Siamenoside I and Their Distributions in Rats.

Siamenoside I is the sweetest mogroside that has several kinds of bioactivities, and it is also a constituent of Siraitiae Fructus, a fruit and herb in China. Hitherto the metabolism of siamenoside I in human or animals remains unclear. To reveal its metabolic pathways, a high-performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrometry (HPLC-ESI-IT-TOF-MS(n)) method was used to profile and identify its metabolites in rats. Altogether, 86 new metabolites were identified or tentatively identified, and 23 of them were also new metabolites of mogrosides. In rats, siamenoside I was found to undergo deglycosylation, hydroxylation, dehydrogenation, deoxygenation, isomerization, and glycosylation reactions. Among them, deoxygenation, pentahydroxylation, and didehydrogenation were novel metabolic reactions of mogrosides. The distributions of siamenoside I and its 86 metabolites in rat organs were firstly reported, and they were mainly distributed to intestine, stomach, kidney, and brain. The most widely distributed metabolite was mogroside IIIE. In addition, eight metabolites were bioactive according to literature. These findings would help to understand the metabolism and effective forms of siamenoside I and other mogrosides in vivo.

Identification of miRNAs and their target genes in developing maize ears by combined small RNA and degradome sequencing.

BACKGROUND: In plants, microRNAs (miRNAs) are endogenous ~22 nt RNAs that play important regulatory roles in many aspects of plant biology, including metabolism, hormone response, epigenetic control of transposable elements, and stress response. Extensive studies of miRNAs have been performed in model plants such as rice and Arabidopsis thaliana. In maize, most miRNAs and their target genes were analyzed and identified by clearly different treatments, such as response to low nitrate, salt and drought stress. However, little is known about miRNAs involved in maize ear development. The objective of this study is to identify conserved and novel miRNAs and their target genes by combined small RNA and degradome sequencing at four inflorescence developmental stages. RESULTS: We used deep-sequencing, miRNA microarray assays and computational methods to identify, profile, and describe conserved and non-conserved miRNAs at four ear developmental stages, which resulted in identification of 22 conserved and 21-maize-specific miRNA families together with their corresponding miRNA*. Comparison of miRNA expression in these developmental stages revealed 18 differentially expressed miRNA families. Finally, a total of 141 genes (251 transcripts) targeted by 102 small RNAs including 98 miRNAs and 4 ta-siRNAs were identified by genomic-scale high-throughput sequencing of miRNA cleaved mRNAs. Moreover, the differentially expressed miRNAs-mediated pathways that regulate the development of ears were discussed. CONCLUSIONS: This study confirmed 22 conserved miRNA families and discovered 26 novel miRNAs in maize. Moreover, we identified 141 target genes of known and new miRNAs and ta-siRNAs. Of these, 72 genes (117 transcripts) targeted by 62 differentially expressed miRNAs may attribute to the development of maize ears. Identification and characterization of these important classes of regulatory genes in maize may improve our understanding of molecular mechanisms controlling ear development.

Multiple factors influenced the aggregation behavior of adult Eucryptorrhynchus scrobicuatus (Coleoptera: Curculionidae).

Eucryptorrhynchus scrobiculatus (Motschulsky) (Coleoptera: Curculionidae) is a notorious pest of Ailanthus altissima (Mill.) Swingle (Sapindales: Simaroubaceae). E. scrobiculatus adults typically aggregate under leaves and in soil crevices at the base of A. altissima in the field. We hypothesize that the environmental factors and conspecific signals determine their aggregation behavior. To test this, we investigated adult numbers in light-exposed and shaded areas of the sample trees and conducted experiments in both field and lab settings. Results revealed that (i) greater adult distribution in shaded areas; (ii) significant influence of temperature and illumination on aggregation tendency in the field; (iii) no gender-based difference in aggregation degree and maximum aggregation between light and dark; (iv) the host plant triggering the aggregation tendency, negatively affected in the absence; (v) the aggregation tendency of E. scrobiculatus weakened with the temperature gradually changing to ordinary temperature; and (vi) mutual attraction and chemical attraction between males and females. Thus, the aggregation behavior was influenced by factors including temperature, light intensity, host plant, and conspecific signals, but light's role was not obvious in the lab.

Cocatalyst Modified Polymeric Carbon Nitride Photoanode for Enhanced Photoelectrochemical Properties.

As a new organic photocatalyst, polymeric carbon nitride (CN) has shown good application potential in the field of photoelectrochemistry due to its unique physical and chemical properties, but its application has been seriously hindered due to its inherent characteristics such as the difficulty in charge separation. In this study, FeOOH modified CN photoanode (CN-Fe) was constructed to investigate the effect of the cocatalyst on the charge injection capacity of organic semiconductor photoelectrodes. The experimental results demonstrate significant improvement in the charge injection efficiency of the photoanode due to the introduction of FeOOH cocatalyst, leading to enhanced photoelectrochemical performance with approximately 2.4 times increase in photocurrent density. By thoroughly investigating the mechanism behind the loading of FeOOH on the polymeric carbon nitride photoanode, we gained profound insights into the behavior of charge carriers and reaction kinetics during the photoelectrocatalytic process.

Synthesis and anti-inflammatory activity of mogrol derivatives modified at C24 site.

Mogrol, the aglycone of well-known sweeter mogrosides, shows potent anti-inflammatory activity. In this study, forty-two mogrol derivatives bearing various pharmacophores with oxygen or nitrogen atoms were designed and synthesized via structural modification at C24 site, and their anti-inflammatory activity were screened against lipopolysaccharide (LPS)-induced RAW264.7 cells. Compared with mogrol, most of derivatives exhibited stronger inhibition of NO production without cytotoxicity. In particular, compound B5 that contained an indole motif effectively suppressed the secretion of inflammatory mediators including TNF-α and IL-6, and inhibited the expression levels of TLR4, p-p65 and iNOS proteins. Molecular docking showed that the active B5 interacted with amino acid residues of iNOS protein through π-π stacking and hydrophobic interactions with binding affinity value of -12.1 kcal/mol, which was much stronger than mogrol (-8.9 kcal/mol). These results suggest that derivative B5 is a promising anti-inflammatory molecule and the strategy of hybridizing indole skeleton on mogrol is worthy for further attention.

Comparative analysis of mesenchymal stem cells from adult mouse adipose, muscle, and fetal muscle.

Recently, increasing evidence supports that adult stem cells are the part of a natural system for tissue growth and repair. This study focused on the differences of mesenchymal stem cells from adult adipose (ADSCs), skeletal muscle (MDSCs) and fetal muscle (FMSCs) in biological characteristics, which is the key to cell therapy success. Stem cell antigen 1 (Sca-1) expression of MDSCs and FMSCs at passage 3 was two times more than that at passage 1 (P < 0.0001). After 28-day myogenic induction, higher expression levels of skeletal muscle-specific genes were observed in MDSCs than FMSCs (P < 0.01), and the lowest expression levels were demonstrated in ADSCs among three cells (P < 0.01). Besides, M-Cad and MyHC expressions in ADSCs were not detected by immunofluorescence or real-time quantitative PCR. Furthermore, after 14 days adipogenic induction, PPARγ2, LPL and aP2 mRNA expressions were higher in ADSCs vs. MDSCs (P < 0.01). Besides, MSCs from adult or fetal muscle expressed higher OCN and OPN than ADSCs after 28 days osteogenic induction (P < 0.01). Taken together, our results suggested that cell source and developmental stage had great impacts on biological properties of mesenchymal stem cells, and proper consideration of all the issues is necessary.

GUESS - A simple approach to accelerate optimization countercurrent separation.

The generally useful estimate of solvent systems (GUESS) method, which is based on thin layer chromatography, is a simple and practical method for selecting solvent systems for countercurrent chromatography (CCC). However, it is rarely used for complex samples derived from natural products. In this study, GUESS was used for CCC solvent system selection and polarity-adjusted CCC separations of several fractions, which were obtained from a silica gel column containing complex compositions with a broad polarity from Salvia bowleyana Dunn. The GUESS method was performed on five fractions based on solvent systems in the n-hexane-ethyl acetate-methanol-water (HEMWat) family. Based on the GUESS results, the optimal solvent systems were selected for CCC separation. Twelve diterpenoids were obtained from the five silica gel column fractions of S. bowleyana Dunn using elution-extrusion countercurrent chromatography (EECCC). These demonstrate that GUESS guidance and the polarity adjustment of the solvent system accelerate the optimization of CCC separation conditions and simplify the process of accommodating a broad polarity of components in complicated mixture fractions. We therefore confirmed the feasibility and advantage of the GUESS method for complex natural chemical component separations.