Genomic variation, environmental adaptation, and feralization in ramie, an ancient fiber crop.

Feralization is an important evolutionary process, but the mechanisms behind it remain poorly understood. Here, we use the ancient fiber crop ramie (Boehmeria nivea (L.) Gaudich.) as a model to investigate genomic changes associated with both domestication and feralization. We first produced a chromosome-scale de novo genome assembly of feral ramie and investigated structural variations between feral and domesticated ramie genomes. Next, we gathered 915 accessions from 23 countries, comprising cultivars, major landraces, feral populations, and the wild progenitor. Based on whole-genome resequencing of these accessions, we constructed the most comprehensive ramie genomic variation map to date. Phylogenetic, demographic, and admixture signal detection analyses indicated that feral ramie is of exoferal or exo-endo origin, i.e., descended from hybridization between domesticated ramie and the wild progenitor or ancient landraces. Feral ramie has higher genetic diversity than wild or domesticated ramie, and genomic regions affected by natural selection during feralization differ from those under selection during domestication. Ecological analyses showed that feral and domesticated ramie have similar ecological niches that differ substantially from the niche of the wild progenitor, and three environmental variables are associated with habitat-specific adaptation in feral ramie. These findings advance our understanding of feralization, providing a scientific basis for the excavation of new crop germplasm resources and offering novel insights into the evolution of feralization in nature.

Anti-inflammatory activity of fluorine-substituted benzo[h]quinazoline-2-amine derivatives as NF-κB inhibitors.

Excessive inflammation can cause loss of tissue or organ function, leading to a number of chronic diseases and sometimes even death. Traditional treatment strategies for inflammation have mainly involved steroidal and non-steroidal anti-inflammatory drugs, but both have increasingly prominent side effects. Nuclear factor kappa B (NF-κB) inhibitors with anti-inflammatory properties and low toxicity are a new therapeutic strategy for the treatment of inflammatory diseases. To obtain novel NF-κB inhibitors, a series of 3,4-dihydronaphthalen-1(2H)-one derivatives (DHNs 6a-s), 1,4,5,6-tetrahydrobenzo[h]quinazolin-2-amine derivatives (BQAs 7a-c) and 5,6-dihydrobenzo[h]quinazolin-2-amine derivatives (BQAs 8a-p) were designed and synthesized, and characterized by NMR and HRMS. By evaluating toxicity and anti-inflammatory properties, fluorine-substituted 8c showed more potential anti-inflammatory activity and lower toxicity. 8c significantly reduced the phosphorylation of IκBα and p65, thereby inhibiting the NF-κB signaling pathway. In addition, 8c markedly decreased reactive oxygen species (ROS) production and downregulated the expression of NOD-like receptor pyrin domain-containing protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC) and cysteine aspartate protein hydrolase-1 (caspase-1). Therefore, compound 8c is expected to be a candidate compound for NF-κB inhibition and deserves further research and development.

Synthesis, biological evaluation and molecular docking studies of novel diosgenin derivatives as anti-inflammatory agents.

Thirty-two novel DG F-spiroacetal ring-opening derivatives, including 24 acetylated derivatives and 8 nitrogenous derivatives, were designed and synthesized from diosgenin (DG). The cytotoxicity of the novel derivatives was evaluated by MTT assay, except for compounds 4a, 4e, 4i, 4 l, 5a and 5 h, which were potentially cytotoxic to RAW264.7 cells, all the other derivatives had no significant cytotoxicity. The NO release inhibitory activities of novel derivatives were screened by Griess method. The results showed that the anti-inflammatory activity of the DG acetylated derivatives was stronger than the nitrogenous derivatives, and 4a-4 m containing acetyl groups at the 3-position may have better anti-inflammatory effects than 5a-5 k containing free hydroxyl groups. In ELISA assay, compound 4 m exhibited potent anti-inflammatory activity by inhibiting the production of NO in RAW264.7 cells activated by LPS with IC50 values 0.449 ± 0.050 µM. The results of docking experiments showed that 4 m has a good affinity for p65 protein.

Anti-neuroinflammatory effects of novel 5,6-dihydrobenzo[h]quinazolin-2-amine derivatives in lipopolysaccharide-stimulated BV2 microglial cells.

Neuroinflammation is an intricate process that is associated with both normal and pathological conditions. Microglia-mediated neuroinflammation is known to lead to various neurodegenerative and neurological disorders. A series of 3,4-dihydronaphthalen-1(2H)-one derivatives (1-15) and novel 5,6-dihydrobenzo[h]quinazolin-2-amine derivatives (16-30) were synthesized and characterized by various analytical methods, such as NMR and HRMS. All compounds were evaluated for toxicity, screened for their anti-neuroinflammatory properties, and investigated for the potential molecular mechanism of lipopolysaccharide (LPS) induction in BV2 microglia. Structure activity relationship analysis showed that compound 17 substituted by the 7-fluorine atom on the A-ring and the 3-methoxy on the D-ring had more potential anti-neuroinflammatory activity by inhibiting the secretion of cytokines TNF-α and IL-6. The results of western blotting assay showed that 17 significantly blocked the activation and phosphorylation of IκBα, significantly reduce the expression of NLRP3 inflammatory vesicle-associated proteins, and thus inhibit the activation of NF-κB pathway. Thus, compound 17 was demonstrated to be an excellent potential therapeutic agent for the treatment of neuroinflammation-related diseases.

Clone and Function Verification of the OPR gene in Brassica napus Related to Linoleic Acid Synthesis.

BACKGROUND: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids (FA), and each fatty acid was regulated by different genes. Furthermore, different FA affect each other, which needs continuous and in-depth research to obtain more clear results in Brassica napus. RESULTS: In this paper, broad-scale miRNA expression profiles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were verified using a quantitative real-time PCR (RT-qPCR) analysis, we found that the target gene of bna-miR156b > c > g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35 s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T1 and T2 generation, the content of linoleic acid (LA) increased significantly in OE but deceased in OPRi. CONCLUSIONS: This is the first study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid profiles in oilseed for breeding programs.

Comparative transcriptomics reveals the selection patterns of domesticated ramie.

Although domestication has dramatically altered the phenotype, physiology, and life history of ramie (Boehmeria nivea) plants, few studies have investigated the effects of domestication on the structure and expression pattern of genes in this fiber crop. To investigate the selective pattern and genetic relationships among a cultivated variety of ramie (BNZ: B. nivea, ZZ1) and four wild species, BNT (B. nivea var. tenacissima), BNN (B. nivea var. nipononivea), BNW (B. nivea var. nivea), and BAN (B. nivea var. viridula), in the section Tilocnide, we performed an RNA sequencing analysis of these ramie species. The de novo assembly of the "all-ramie" transcriptome yielded 119,114 unigenes with an average length of 633 bp, and a total of 7,084 orthologous gene pairs were identified. The phylogenetic tree showed that the cultivar BNZ clustered with BAN in one group, BNW was closely related to BNT, and BNN formed a separate group. Introgression analysis indicated that gene flow occurred from BNZ to BNN and BAN, and between BAN and BNN. Among these orthologs, 2,425 and 269 genes underwent significant purifying and positive selection, respectively. For these positively selected genes, oxidation-reduction process (GO:0055114) and stress response pathways (GO:0006950) were enriched, indicating that modulation of the cellular redox status was important during both ramie fiber evolution and improvement. Two genes related to the suppression of flowering and one gene annotated as a flowering-promoting factor were subjected to positive selection, probably caused by human manipulation. Additionally, five genes were homologs of those involved in abiotic stress tolerance and disease resistance, with higher expression levels in the cultivar BNZ than in the wild species. Collectively, the results of this study indicated that domestication has resulted in the upregulation of many genes involved in the abiotic and biotic stress responses, fiber yield, and plant growth of ramie.

Draft genome sequence of ramie, Boehmeria nivea (L.) Gaudich.

Ramie, Boehmeria nivea (L.) Gaudich, family Urticaceae, is a plant native to eastern Asia, and one of the world's oldest fibre crops. It is also used as animal feed and for the phytoremediation of heavy metal-contaminated farmlands. Thus, the genome sequence of ramie was determined to explore the molecular basis of its fibre quality, protein content and phytoremediation. For further understanding ramie genome, different paired-end and mate-pair libraries were combined to generate 134.31 Gb of raw DNA sequences using the Illumina whole-genome shotgun sequencing approach. The highly heterozygous B. nivea genome was assembled using the Platanus Genome Assembler, which is an effective tool for the assembly of highly heterozygous genome sequences. The final length of the draft genome of this species was approximately 341.9 Mb (contig N50 = 22.62 kb, scaffold N50 = 1,126.36 kb). Based on ramie genome annotations, 30,237 protein-coding genes were predicted, and the repetitive element content was 46.3%. The completeness of the final assembly was evaluated by benchmarking universal single-copy orthologous genes (BUSCO); 90.5% of the 1,440 expected embryophytic genes were identified as complete, and 4.9% were identified as fragmented. Phylogenetic analysis based on single-copy gene families and one-to-one orthologous genes placed ramie with mulberry and cannabis, within the clade of urticalean rosids. Genome information of ramie will be a valuable resource for the conservation of endangered Boehmeria species and for future studies on the biogeography and characteristic evolution of members of Urticaceae.

Development and validation of liquid chromatography-tandem mass spectrometry method for quantification of a potential anticancer triterpene saponin from seeds of Nigella glandulifera in rat plasma: application to a pharmacokinetic study.

Nigella glandulifera Freyn et Sit is a folk medicinal plant, whose seeds show significant anticancer activities attributed to triterpene saponins and volatile oil. In this study, an in vitro cytotoxicity assay demonstrated that Nigella A, the major component of triterpene saponins extracted from N. glandulifera, exhibited growth inhibition in the human lung carcinoma A-549 cell line. Due to this potential activity, a reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify Nigella A in rat plasma for a pharmacokinetic study was developed. Nigella A and pravastatin, as internal standard (IS), were extracted from rat plasma using acetonitrile to precipitate protein. Separation was performed on an Agilent Zorbax SB-Aq (3.0 × 150 mm, 3.5 µm) column using a gradient elution method with acetonitrile-0.1% formic acid in water at a flow rate of 0.35 mL/min. Detection was performed using an electrospray ionization in a negative ion multiple reaction monitoring mode. The deprotonated precursor to product ion transitions monitored for Nigella A and IS was at m/z 1352.7→882.6 and m/z 423.1→321.0, respectively. The linear range was 0.240-120 µg/mL with a square regression coefficient (r=0.9996). The intra-day and inter-day precision was less than 6.93%. The simple extraction procedure provided recovery ranged from 92.32 to 95.44% for both analyte and IS. The method was proved to be reliable, precise, and accurate, and was successfully applied to a pharmacokinetic study of Nigella A in rats after i.v. administration via the tail vein at doses of 10, 20, and 30 mg/kg.

Development of a core collection for ramie by heuristic search based on SSR markers.

There are more than 2000 ramie germplasms in the National Ramie Germplasm Nursery affiliated with the Institute of Bast Fiber Crops, Chinese Academy of Agricultural Science, China. As it is difficult to perform effective conservation, management, evaluation, and utilization of redundant genetic resources, it is necessary to construct a core collection by using molecular markers. In this study, a core collection of ramie consisting of 22 germplasms was constructed from 108 accessions by heuristic search based on 21 Simple Sequence Repeat (SSR) marker combinations. The results showed that there is a poor relationship between the core collection and the geographic distribution. The number of amplification bands for the core collection was the same as that for the entire collection. Shannon's index for three of the SSR primers (14%) and Nei's index for nine of the SSR primers (19%) were lower in the core collection than in the entire collection. The true core collection had wider genetic diversity compared with the random core collection. Collectively, the core collection constructed in this study is reliable and represents the genetic diversity of all the 108 accessions.

Study on antitubercular constituents from the seeds of Nigella glandulifera.

Two new compounds with five known compounds have been isolated from EtOH extract of the seeds of Nigella glandulifera. On the basis of their spectroscopic and chemical evidence, the new compounds were elucidated as methoxynigeglanine (1) and 6-methoxythymol-3-O-ß-D-glucopyranoside (4). Compounds 1-4 showed moderate antitubercular activity against Mycobacterium tuberculosis strain H37Rv with minimal inhibitory concentration (MIC) values of 32-250 µg/mL.

Aa TFL1 confers an age-dependent response to vernalization in perennial Arabis alpina.

Flowering of many plants is induced by environmental signals, but these responses can depend on the age of the plant. Exposure of Arabidopsis thaliana to vernalization (winter temperatures) at germination induces flowering, whereas a close perennial relative Arabis alpina only responds if exposed when at least 5 weeks old. We show that vernalization of these older A. alpina plants reduces expression of the floral repressor PEP1 and activates the orthologs of the Arabidopsis flowering genes SOC1 (Aa SOC1) and LFY (Aa LFY). By contrast, when younger plants are vernalized, PEP1 and Aa SOC1 mRNA levels change as in older plants, but Aa LFY is not expressed. We demonstrate that A. alpina TFL1 (Aa TFL1) blocks flowering and prevents Aa LFY expression when young plants are exposed to vernalization. In addition, in older plants, Aa TFL1 increases the duration of vernalization required for Aa LFY expression and flowering. Aa TFL1 has similar functions in axillary shoots, thus ensuring that following a flowering episode vegetative branches are maintained to continue the perennial life cycle. We propose that Aa TFL1 blocks flowering of young plants exposed to vernalization by setting a threshold for a flowering pathway that is increased in activity as the shoot ages, thus contributing to several perennial traits.

[Surveillance on Rickettsia in epidemic areas of scrub typhus in Xinyang areas of Henan].

OBJECTIVE: To understand the epidemic status of Rickettsia in Xinyang areas of Henan province. METHODS: Samples including liver, spleen, kidney from mouse and chigger mites from Xinyang areas and serum samples were detected by nested-polymerase chain reaction (PCR) and indirect immunofluorescence assay (IFA). RESULTS: In 62 viscus samples from mice organs, the positive rates were 16.13%, 8.06% and 6.45% for Orientia tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. In blood clots samples from mice, the positive rates were 8.06%, 6.45% and 1.61 % for O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae respectively. Three out of 26 mouse serum samples were positive for the predicted fluorexcent intensity O. tsutsugamushi. CONCLUSION: Using nested-PCR and IFA methods, O. tsutsugamushi, R. typhii and Spotted fever group rickettsiae were detected in the captured mice living in Xinyang areas of Henan province. Results showed that there were intensive natural reserviors of Rickettsia in Henan province, suggesting that the risk of outbreak of Rickettsia in these areas was high.

Molecular epidemic survey on co-prevalence of scrub typhus and marine typhus in Yuxi city, Yunnan province of China.

BACKGROUND: Human rickettsioses are worldwide zoonoses and it is not easy to differentiate them from other infectious diseases because of their atypical manifestation. In recent years the number of patients with fever of unknown causes from Hongta District CDC, Yuxi city of Yunnan Province has been increasing significantly in the summer. Diagnosis of scrub typhus was made by local clinicians. In order to ascertain the disease, we undertook a laboratory investigation for such patients from August 18 to 26, 2005. METHODS: Active surveillance was conducted by Hongta District CDC Yuxi city of Yunnan Province from 2002 to 2004 and basic data were obtained from cases confirmed according to clinical definitions. Average incidences and town-level incidences were calculated during the study periods. Blood samples were analyzed by PCR and serological test. Based on the groEL gene sequences a paired general outer primers (Gro-1 and Gro-2) targeting typhus, spotted fever as well as scrub typhus and two paired inner primers (SF1, SR2 and TF1, TR2) for typhus together with spotted fever and scrub typhus, respectively, were designed to perform a multiplex-nested PCR. Serological assay was carried out by indirect immunofluorescence assay with 7 different rickettsial antigens, i.e., R.mossori, R.sibirica, R.conorii, O.tsutsugamushi, B.quintana, B.henselae and Coxilella burnetii phase II Ag. RESULTS: Epidemiological surveillance showed that from 2002 to 2004, the average incidences of the scrub typhus or scrub typhus with murine typhus were 222.1/10(5), 204.3/10(5) and 109.6/10(5), respectively. Of 13 blood samples taken during acute stage of illness, 6 showed the amplified products for scrub typhus and the sequenced products showed 100%, 99%, 99%, 99%, 99%, 99% similarity to O.tsutsugamushi Karp but they shared the same deduced amino acid sequences, which indicated 100% identity with the heat shock protein of the O.tsutsugamushi Karp strain. Five yielded PCR products for murine typhus and their corresponding nucleotide sequences exhibited 100%, 100%, 99%, 99% and 99% similarity to R. mossori Wilmington and the analyses of predicted amino acid sequences indicated 100%, 100%, 98%, 98% and 98% identity with the heat shock protein of R. mossori Wilmington strain. Of the 8 PCR positive patients, 3 showed a co-infection of scrub typhus with murine typhus. All the 13 serum samples from febrile patients were positive against O. tsutsugamushi and 8 of them were positive against R. mossori. All of the 8 paired specimens had four-fold elevation of antibody against O. tsutsugamushi, and seroconversion for typhus was demonstrated in 3 paired serum samples. Another finding in the study was that a high seropositive prevalence (76.9%) of Q fever was detected. CONCLUSION: It's confirmed that co-prevalence of scrub typhus with murine typhus are occurring in Yuxi city of Yunnan province, China. Other rickettsial diseases also need to be investigated in these areas.