Publications by authors named "Jinfeng Hou"

23 Publications

  • Page 1 of 1

Mechanism of genome instability mediated by human DNA polymerase mu misincorporation.

Nat Commun 2021 06 18;12(1):3759. Epub 2021 Jun 18.

Institute of Biophysics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.

Pol μ is capable of performing gap-filling repair synthesis in the nonhomologous end joining (NHEJ) pathway. Together with DNA ligase, misincorporation of dGTP opposite the templating T by Pol μ results in a promutagenic T:G mispair, leading to genomic instability. Here, crystal structures and kinetics of Pol μ substituting dGTP for dATP on gapped DNA substrates containing templating T were determined and compared. Pol μ is highly mutagenic on a 2-nt gapped DNA substrate, with T:dGTP base pairing at the 3' end of the gap. Two residues (Lys438 and Gln441) interact with T:dGTP and fine tune the active site microenvironments. The in-crystal misincorporation reaction of Pol μ revealed an unexpected second dGTP in the active site, suggesting its potential mutagenic role among human X family polymerases in NHEJ.
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http://dx.doi.org/10.1038/s41467-021-24096-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213813PMC
June 2021

Characterization and transcriptomic analysis of a novel yellow-green leaf wucai (Brassica campestris L.) germplasm.

BMC Genomics 2021 Apr 12;22(1):258. Epub 2021 Apr 12.

College of Horticulture, Vegetable Genetics and Breeding Laboratory, Anhui Agricultural University, 130 West Changjiang Road, Hefei, 230036, Anhui, China.

Background: Leaf color mutants are the ideal materials to explore the pathways of chlorophyll (Chl) metabolism, chloroplast development, and photosynthesis system. In this study, a spontaneous yellow-green leaf wucai (Brassica campestris L.) mutant "WY16-13" was identified, which exhibited yellow-green leaf color during its entire growth period. However, current understanding of the molecular mechanism underlying Chl metabolism and chloroplast development of "WY16-13" is limited.

Results: Total Chl and carotenoid content in WY16-13 was reduced by 60.92 and 58.82%, respectively, as compared with its wild type parental line W16-13. Electron microscopic investigation revealed fewer chloroplasts per cell and looser stroma lamellae in WY16-13 than in W16-13. A comparative transcriptome profiling was performed using leaves from the yellow-green leaf type (WY16-13) and normal green-leaf type (W16-13). A total of 54.12 million (M) (WY16-13) and 56.17 M (W16-13) reads were generated. A total of 40,578 genes were identified from the mapped libraries. We identified 3882 differentially expressed genes (DEGs) in WY16-13 compared with W16-13 (i.e., 1603 upregulated genes and 2279 downregulated genes). According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, these DEGs are involved in porphyrin and Chl metabolism [i.e., chlorophyllase (CLH), heme oxygenase (HO), chlorophyll (ide) b reductase (NYC), and protochlorophyllide oxidoreductase (POR) genes], carbohydrate metabolism, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, deficiency in Chl biosynthetic intermediates in WY16-13 revealed that the formation of the yellow-green phenotype was related to the disorder of heme metabolism.

Conclusions: Our results provide valuable insights into Chl deficiency in the yellow-green leaf mutant and a bioinformatics resource for further functional identification of key allelic genes responsible for differences in Chl content.
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http://dx.doi.org/10.1186/s12864-021-07573-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040211PMC
April 2021

Impact of overweight/obesity on clinical outcomes after reduction for intussusception in children.

Pediatr Surg Int 2021 Jul 7;37(7):887-895. Epub 2021 Apr 7.

Department of Neonatal Gastrointestinal Surgery, Children's Hospital of Chongqing Medical University, National Clinical Research Center for Child Health and Disorders, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing, 400014, China.

Purpose: Childhood obesity is a worsening epidemic. Little is known about the impact of overweight and obesity (OV-OB) on clinical outcomes after reduction for intussusception in children. The aim of this study was to compare clinical outcomes after primarily air enema reduction for intussusception in grouped OV-OB (body mass index-for-age percentile ≥ 85) pediatric patients compared with no OV-OB patients.

Methods: A retrospective study of 564 children who had undergone intussusception reduction via pneumatic reduction (PR) from April 2018 to January 2020 was conducted with assessments of demographic data, clinical symptoms, pre-reduction examination, and reduction results. One-to-one propensity score matching (PSM) was performed to compare clinical outcomes between patients with and without OV-OB, and the risk factors affecting recurrence and surgical reduction were analyzed in the PSM population.

Results: Of the 564 patients, 132 cases (23.4%) were OV-OB (overweight: 95 cases; obesity: 37 cases). In the propensity-matched analysis, the OV-OB group showed a significant increase in surgical reduction (10.2% versus 0.9%, P = 0.005) and recurrence (47.2% versus 10.2%, P < 0.001), excretion time of carbon powder after PR (median: 11.2 h versus 8.4 h, P < 0.001), higher maximum pressure of PR (median: 10.2 kPa versus 7.8 kPa, P < 0.001), and number of PR attempts (mean: 2.0 versus 1.4, P < 0.001). There were no significant differences in the reasons for surgery (PR failure or bowel perforation), time to recurrence (early or late), and the times of recurrences ≥ 2. After applying the multivariate logistic regression analysis, we found that OV-OB and white blood cell count ≥ 20 × 10/L were risk factors for both surgical reduction and the recurrence of intussusception.

Conclusion: This study suggested that childhood OV-OB was associated with the failure of PR and recurrence of intussusception after reduction, which should be paid more attention in clinical practice.
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http://dx.doi.org/10.1007/s00383-021-04888-0DOI Listing
July 2021

The effect of exogenous 24-epibrassinolide pretreatment on the quality, antioxidant capacity, and postharvest life of wucai ( L.).

Food Sci Nutr 2021 Mar 23;9(3):1323-1335. Epub 2021 Jan 23.

College of Horticulture Vegetable Genetics and Breeding Laboratory Anhui Agricultural University Hefei China.

The quality of green leafy vegetables is easily lost during the postharvest period. The effect of exogenous 24-epibrassinolide (EBR) pretreatment on the quality of wucai was evaluated in the present study. Wucai plants were sprayed twice with 0.1 μM EBR before harvesting. Two storage temperatures were tested: 25°C and 4°C. At 4°C, EBR pretreatment significantly delayed the degradation of the pigment and plant water loss. Furthermore, we measured the activity of key enzymes of the ascorbic acid (AsA)-glutathione (GSH) cycle, the content of the main metabolites, and the expression of the AsA metabolism-related genes in leaves. The results indicated that all three plants showed stronger antioxidant capacity after EBR pretreatment. At 4°C and 25°C, the storage time of wucai was 20 days and 7 days after EBR treatment, while the samples could be stored for 14 days and 4 days without EBR treatment application, respectively. At 4°C, the nutritional properties of wucai pretreated with EBR, such as total free amino acids, total soluble sugar, and cellulose contents, were higher than those of the control, while the content of nitrite and lignin was lower than that of the control. Hence, EBR pretreatment was able to enhance the antioxidant capacity of wucai, maintain normal leaf color and shape during storage, and delay the decline of nutritional properties; therefore, EBR pretreatment has potential commercial value for prolonging the market life of wucai.
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http://dx.doi.org/10.1002/fsn3.2075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958569PMC
March 2021

MazEF Toxin-Antitoxin System-Mediated DNA Damage Stress Response in .

Front Genet 2021 19;12:632423. Epub 2021 Feb 19.

Institute of Biophysics, College of Life Sciences, Zhejiang University, Hangzhou, China.

shows marked resistance to various types of DNA-damaging agents, including mitomycin C (MMC). A type II toxin-antitoxin (TA) system that responds to DNA damage stress was identified in , comprising the toxin MazF-dr and the antitoxin MazE-dr. The cleavage specificity of MazF-dr, an endoribonuclease, was previously characterized. Here, we further investigated the regulatory role of the MazEF system in the response to DNA damage stress in . The crystal structure of MazF (MazF-dr) was determined at a resolution of 1.3 Å and is the first structure of the toxin of the TA system of . MazF-dr forms a dimer mediated by the presence of interlocked loops. Transcriptional analysis revealed 650 downregulated genes in the wild-type (WT) strain, but not in the mutant strain, which are potentially regulated by MazEF-dr in response to MMC treatment. Some of these genes are involved in membrane trafficking and metal ion transportation. Subsequently, compared with the WT strain, the mutant strain exhibited much lower MMC-induced intracellular iron concentrations, reactive oxygen species (ROS), and protein carbonylation levels. These results provide evidence that MazEF-mediated cell death in might be caused by an increase in ROS accumulation upon DNA damage stress.
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http://dx.doi.org/10.3389/fgene.2021.632423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7933679PMC
February 2021

Transcriptome Analysis Reveals the Symbiotic Mechanism of -Induced Gall Formation of .

Mol Plant Microbe Interact 2021 Feb 5;34(2):168-185. Epub 2021 Jan 5.

Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University; Hefei 230036, China.

is a perennial aquatic vegetable, whose symbiosis with the fungus (member of Basidiomycota, class ) results in the establishment of swollen gall formations. Here, we analyzed symbiotic relations of and , using a triadimefon (TDF) treatment and transcriptome sequencing (RNA-seq). Specifically, accurately identify the whole growth cycle of Microstructure observations showed that the presence of could be clearly observed after gall formation but was absent after the TDF treatment. A total of 17,541 differentially expressed genes (DEGs) were identified, based on the transcriptome. According to gene ontology term and Kyoto Encyclopedia of Genes and Genomes pathway results, plant hormone signal transduction, and cell wall-loosening factors were all significantly enriched due to infecting ; relative expression levels of hormone-related genes were identified, of which downregulation of indole 3-acetic acid (IAA)-related DEGs was most pronounced in JB_D versus JB_B. The ultra-high performance liquid chromatography analysis revealed that IAA, zeatin+trans zeatin riboside, and gibberellin 3 were increased under infection. Based on our results, we proposed a hormone-cell wall loosening model to study the symbiotic mechanism of gall formation after infects . Our study thus provides a new perspective for studying the physiological and molecular mechanisms of infection of causing swollen gall formations as well as a theoretical basis for enhancing future yields of cultivated .[Formula: see text] The author(s) have dedicated the work to the public domain under the Creative Commons CC0 "No Rights Reserved" license by waiving all of his or her rights to the work worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law. 2021.
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http://dx.doi.org/10.1094/MPMI-05-20-0126-RDOI Listing
February 2021

Genome-wide analysis of proline-rich extension-like receptor protein kinase (PERK) in Brassica rapa and its association with the pollen development.

BMC Genomics 2020 Jun 15;21(1):401. Epub 2020 Jun 15.

Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University, Hefei, 230036, China.

Background: Proline-rich extension-like receptor protein kinases (PERKs) are an important class of receptor kinases located in the plasma membrane, most of which play a vital role in pollen development.

Results: Our study identified 25 putative PERK genes from the whole Brassica rapa genome (AA). Phylogenetic analysis of PERK protein sequences from 16 Brassicaceae species divided them into four subfamilies. The biophysical properties of the BrPERKs were investigated. Gene duplication and synteny analyses and the calculation of Ka/Ks values suggested that all 80 orthologous/paralogous gene pairs between B. rapa and A. thaliana, B. nigra and B. oleracea have experienced strong purifying selection. RNA-Seq data and qRT-PCR analyses showed that several BrPERK genes were expressed in different tissues, while some BrPERKs exhibited high expression levels only in buds. Furthermore, comparative transcriptome analyses from six male-sterile lines of B. rapa indicated that 7 BrPERK genes were downregulated in all six male-sterile lines. Meanwhile, the interaction networks of the BrPERK genes were constructed and 13 PERK coexpressed genes were identified, most of which were downregulated in the male sterile buds.

Conclusion: Combined with interaction networks, coexpression and qRT-PCR analyses, these results demonstrated that two BrPERK genes, Bra001723.1 and Bra037558.1 (the orthologs of AtPERK6 (AT3G18810)), were downregulated beginning in the meiosis II period of male sterile lines and involved in anther development. Overall, this comprehensive analysis of some BrPERK genes elucidated their roles in male sterility.
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http://dx.doi.org/10.1186/s12864-020-06802-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296749PMC
June 2020

Comparative Analysis of Rhizosphere Microbiomes of Southern Highbush Blueberry ( L.), Darrow's Blueberry ( Camp), and Rabbiteye Blueberry ( Aiton).

Front Microbiol 2020 12;11:370. Epub 2020 Mar 12.

Department of Cell and Molecular Biology, School of Biological, Environmental, and Earth Sciences, The University of Southern Mississippi, Hattiesburg, MS, United States.

Plants are inhabited by millions of parasitic, commensal, and mutualistic microorganisms that coexist in complex ecological communities, and profoundly affect the plant's productivity, health, and capacity to cope with environmental stress. Therefore, a better understanding of the rhizosphere microbiome may open a yet untapped avenue for the rational exploitation of beneficial plant-microbe interactions in modern agriculture. Blueberries encompass several wild and cultivated species of shrubs of the genus that are native to North America. They are grown commercially for the production of fruits, which are considered a health food due to the rich content of minerals, trace elements, and phenolic compounds with antioxidant, antitumor, and anti-inflammatory properties. Despite a long history of breeding and extensive commercial use, remarkably little is known about the composition and function of the blueberry root microbiome. To address this gap, we employed molecular approaches to characterize and compare microbial communities inhabiting the roots of rabbiteye blueberry (), Darrow's blueberry (), and southern highbush blueberry (SHB; an interspecific hybrid of and ). Our results revealed that these plant species share a common core rhizobiome, but at the same time differ significantly in the diversity, relative abundance, richness, and evenness of multiple groups of prokaryotic and eukaryotic microorganisms. Although the host signature effects were especially pronounced at the plant species level, we also observed genotype-level variations in the distribution of specific microbial taxa, which suggests that the assembly of the blueberry microbiome is shaped by the plant genotype and modifications associated with the domestication and breeding of members of the genus. We also demonstrated that the studied species differ in the abundance of beneficial rhizobacteria and ericoid mycorrhizal fungi, which play a vital role in their adaptation to soils with low pH and slow turnover of organic matter.
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http://dx.doi.org/10.3389/fmicb.2020.00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7081068PMC
March 2020

Response of photosynthetic capacity and antioxidative system of chloroplast in two wucai ( L.) genotypes against chilling stress.

Physiol Mol Biol Plants 2020 Feb 1;26(2):219-232. Epub 2020 Jan 1.

1Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University, Hefei, 230036 China.

Chilling stress during the growing season could cause a series of changes in wucai ( L.). WS-1 (chilling-tolerant genotype) and Ta2 (chilling-sensitive genotype) were sampled in present study to explore the chilling tolerance mechanisms. Our results indicated that photosynthetic parameters exhibited lower level in Ta2 than in WS-1 under chilling stress. The rapid chlorophyll fluorescence dynamics curve showed that chilling resulted in a greater inactivation of photosystem II reaction center in Ta2. Reactive oxygen species and malondialdehyde content of chloroplast in Ta2 were higher than WS-1. The ascorbate-glutathione cycle in chloroplast of WS-1 played a more crucial role than Ta2, which was confirmed by higher activities of antioxidant enzymes including Ascorbate peroxidase, Glutathione reductase, Monodehydroascorbate reductase and Dehydroascorbate reductase and higher content of AsA and GSH. In addition, the ultrastructure of chloroplasts in Ta2 was more severely damaged. After low temperature stress, the shape of starch granules in Ta2 changed from elliptical to round and the volume became larger than that of WS-1. The thylakoid structure of Ta2 also became dispersed from the original tight arrangement. Combined with our previous study under heat stress, WS-1 can tolerant both chilling stress and heat stress, which was partly due to a stable photosynthetic system and the higher active antioxidant system in plants, in comparison to Ta2.
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http://dx.doi.org/10.1007/s12298-019-00743-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036399PMC
February 2020

Comparative Proteomics Reveals Cold Acclimation Machinery Through Enhanced Carbohydrate and Amino Acid Metabolism in Wucai ( L.).

Plants (Basel) 2019 Nov 6;8(11). Epub 2019 Nov 6.

Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University, Hefei 230036, China.

Limited information is available on the cold acclimation of non-heading Chinese cabbage (NHCC) under low temperatures. In this study, the isobaric tags for relative and absolute quantification (iTRAQ) were used to illustrate the molecular machinery of cold acclimation. Compared to the control (Cont), altogether, 89 differentially expressed proteins (DEPs) were identified in wucai leaves responding to low temperatures (LT). Among these proteins, 35 proteins were up-regulated ((and 54 were down-regulated). These differentially expressed proteins were categorized as having roles in carbohydrate metabolism, photosynthesis and energy metabolism, oxidative defense, amino acid metabolism, metabolic progress, cold regulation, methylation progress, and signal transduction. The fructose, glucose, and sucrose were dramatically increased in response to cold acclimation. It was firstly reported that aspartate, serine, glutamate, proline, and threonine were significantly accumulated under low temperatures. Results of quantitative real-time PCR analysis of nine DEPs displayed that the transcriptional expression patterns of six genes were consistent with their protein expression abundance. Our results demonstrated that wucai acclimated to low temperatures through regulating the expression of several crucial proteins. Additionally, carbohydrate and amino acid conversion played indispensable and vital roles in improving cold assimilation in wucai.
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http://dx.doi.org/10.3390/plants8110474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918420PMC
November 2019

Transcriptome analysis reveals a positive effect of brassinosteroids on the photosynthetic capacity of wucai under low temperature.

BMC Genomics 2019 Nov 6;20(1):810. Epub 2019 Nov 6.

College of Horticulture, Vegetable Genetics and Breeding Laboratory, Anhui Agricultural University, 130 West Changjiang Road, Hefei, 230036, Anhui, China.

Background: Brassinosteroids (BRs) have a positive effect on many processes during plant growth and development, and in response to various abiotic stressors. Low-temperature (LT) stress constricts the geographic distribution, growth, and development of wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). However, there is little information on the global gene expression of BRs under LT stress in wucai. In this study, the molecular roles of 24-epibrassinolide (EBR) after exogenously application, were explored by RNA sequencing under LT conditions.

Results: According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, photosynthesis was significantly enriched after spraying EBR under LT. The transcripts encoding the photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin were up-regulated after the application of EBR. Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. Notably, transcriptional and physiological analyses revealed that under LT stress, plant responses to EBR involved a major reorientation of photosynthesis, as well as porphyrin and chlorophyll metabolism.

Conclusion: This study explored the role of EBR as an LT stress tolerance mechanism in wucai. At the transcription level, LT tolerance manifests as an enhancement of photosynthesis, and the amelioration of porphyrin and chlorophyll metabolism.
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http://dx.doi.org/10.1186/s12864-019-6191-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6836548PMC
November 2019

Physiological and Transcriptomic Analyses Elucidate That Exogenous Calcium Can Relieve Injuries to Potato Plants ( L.) under Weak Light.

Int J Mol Sci 2019 Oct 16;20(20). Epub 2019 Oct 16.

College of Horticulture, Vegetable Genetics and Breeding Laboratory, Anhui Agricultural University, Hefei 230036, China.

Light is one of the most important abiotic factors for most plants, which affects almost all growth and development stages. In this study, physiological indicators suggest that the application of exogenous Ca improves photosynthesis and changes phytohormone levels. Under weak light, photosynthetic parameters of the net photosynthetic rate (P), stomatal conductance (Gs), and transpiration rate (Tr) decreased; the antioxidation systems peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) reduced; the degrees of malondialdehyde (MDA), HO, and superoxide anion (O) free radical damage increased; while exogenous Ca treatment was significantly improved. RNA-seq analysis indicated that a total of 13,640 differently expressed genes (DEGs) were identified and 97 key DEGs related to hormone, photosynthesis, and calcium regulation were differently transcribed. Gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses, plant hormone signal transduction, photosynthesis, carbon metabolism, and phenylpropanoid biosynthesis were significantly enriched. Additionally, quantitative real-time PCR (qRT-PCR) analysis confirmed some of the key gene functions in response to Ca. Overall, these results provide novel insights into the complexity of Ca to relieve injuries under weak light, and they are helpful for potato cultivation under weak light stress.
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http://dx.doi.org/10.3390/ijms20205133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829426PMC
October 2019

Comparative Proteomics Indicates That Redox Homeostasis Is Involved in High- and Low-Temperature Stress Tolerance in a Novel Wucai ( L.) Genotype.

Int J Mol Sci 2019 Aug 1;20(15). Epub 2019 Aug 1.

College of Horticulture, Vegetable Genetics and Breeding Laboratory, Anhui Agricultural University, 130 West Changjiang Road, Hefei 230036, China.

The genotype WS-1, previously identified from novel wucai germplasm, is tolerant to both low-temperature (LT) and high-temperature (HT) stress. However, it is unclear which signal transduction pathway or acclimation mechanisms are involved in the temperature-stress response. In this study, we used the proteomic method of tandem mass tag (TMT) coupled with liquid chromatography-mass spectrometry (LC-MS/MS) to identify 1022 differentially expressed proteins (DEPs) common to WS-1, treated with either LT or HT. Among these 1022 DEPs, 172 were upregulated in response to both LT and HT, 324 were downregulated in response to both LT and HT, and 526 were upregulated in response to one temperature stress and downregulated in response to the other. To illustrate the common regulatory pathway in WS-1, 172 upregulated DEPs were further analyzed. The redox homeostasis, photosynthesis, carbohydrate metabolism, heat-shockprotein, and chaperones and signal transduction pathways were identified to be associated with temperature stress tolerance in wucai. In addition, overexpressed in exhibited higher reduced glutathione (GSH) content and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and less oxidative damage under temperature stress. This result is consistent with the dynamic regulation of the relevant proteins involved in redox homeostasis. These data demonstrate that maintaining redox homeostasis is an important common regulatory pathway for tolerance to temperature stress in novel wucai germplasm.
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http://dx.doi.org/10.3390/ijms20153760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696267PMC
August 2019

Overexpression of a soybean YABBY gene, GmFILa, causes leaf curling in Arabidopsis thaliana.

BMC Plant Biol 2019 Jun 3;19(1):234. Epub 2019 Jun 3.

National Center for Soybean Improvement, National Key Laboratory of Crop Genetics and Germplasm Enhancement, Jiangsu Collaborative Innovation Center for Modern Crop Production, Nanjing Agricultural University, Nanjing, 210095, China.

Background: YABBY genes play important roles in the growth and polar establishment of lateral organs such as leaves and floral organs in angiosperms. However, the functions of YABBY homologous genes are largely unknown in soybean.

Results: In this study, we identified GmFILa encoding a YABBY transcription factor belonging to FIL subfamily. In situ mRNA hybridization analysis indicated that GmFILa had specific expression patterns in leaf as well as in flower bud primordia. Ectopic expression of GmFILa in Arabidopsis thaliana altered the partial abaxialization of the adaxial epidermises of leaves. Besides, GmFILa transgenic plants also exhibited longer flowering period and inhibition of shoot apical meristem (SAM) development compared to the wild type plants. Digital expression data and quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that the expression of GmFILa was induced by biotic and abiotic stresses and hormone treatments. Transcriptome analysis suggested that overexpressing GmFILa yielded 82 significant differentially expressed genes (DEGs) in Arabidopsis leaves, which can be classified into transcription factors, transporters, and genes involved in growth and development, metabolism, signal transduction, redox reaction and stress response.

Conclusions: These results not only demonstrate the roles of GmFILa involved in leaf adaxial-abaxial polarity in Arabidopsis, but also help to reveal the molecular regulatory mechanism of GmFILa based on the transcriptomic data.
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http://dx.doi.org/10.1186/s12870-019-1810-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6547562PMC
June 2019

Comparative Proteomic Analysis Reveals That Chlorophyll Metabolism Contributes to Leaf Color Changes in Wucai ( Brassica campestris L.) Responding to Cold Acclimation.

J Proteome Res 2019 06 8;18(6):2478-2492. Epub 2019 May 8.

College of Horticulture, Vegetable Genetics and Breeding Laboratory , Anhui Agricultural University , 130 West Changjiang Road , Hefei , Anhui 230036 , China.

Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai ( Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7-2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7-1 was selected as the control to evaluate the chlorophyll metabolism changes of W7-2. Compared to W7-1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7-2 were significantly reduced at five developmental stages (13, 21, 29, 37, and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7-2 and W7-1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W7-2 compared with W7-1, respectively. The expression analysis of genes of three subunits of Mg-chelatase ( CHLI1, CHLD, and CHLH), Genomes Uncoupled 4 ( GUN4), and Thioredoxin ( TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7-2. In particular, PORB showed significant up-regulation in W7-2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7-2 and W7-1 were in terms of altered photosynthesis, carbohydrate, and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7-2 than in W7-1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation.
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http://dx.doi.org/10.1021/acs.jproteome.9b00016DOI Listing
June 2019

[Reversed-phase liquid chromatography separation and mass spectrometry fragmentation analysis of mitiglinide and three isomers].

Se Pu 2019 Apr;37(4):412-417

Beijing Institute for Drug Control, Beijing Municipal Key Laboratory for Analysis of Traditional Chinese Medicines and Biological Evaluation, Beijing 102206, China.

The separation of mitiglinide and its three isomer impurities was achieved by reversed-phase ultra-performance liquid chromatography-high resolution mass spectrometry. An ACQUITY UPLC HSS T3 column (100 mm×2.1 mm, 1.8 μm) was used as the stationary phase and water-acetonitrile--pentanol (75:25:1, v/v/v; formic acid was added to adjust pH to 1.8) was used as the mobile phase with a flow rate of 0.4 mL/min. According to the exact mass and high resolution mass spectrometry fragmentation (Q Exactive), significant differences were observed in the fragment ion abundance in the secondary mass spectra of mitiglinide and its three isomer impurities. Two of these isomer impurities were newly discovered. The possible fragmentation mechanisms of mitiglinide and its three isomer impurities were also deduced. The limit of detection of the developed method was 1 μg/L. The linearity of the developed method was good from the limit of quantitation (2 μg/L) to 10000 μg/L with a correlation coefficient of 0.9999. The relative standard deviation of the peak area was 2.0%. On the basis of these results, the sources of the mitiglinide isomer impurities were discussed. Isomer impurity 1 was degraded at high temperature, while isomer impurities 2 and 3 were determined to be synthetic impurities. In addition, samples of mitiglinide calcium raw materials were analyzed.
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http://dx.doi.org/10.3724/SP.J.1123.2018.11046DOI Listing
April 2019

Transgenic Wucai (Brassica campestris L.) produced via Agrobacterium-mediated anther transformation in planta.

Plant Cell Rep 2019 May 13;38(5):577-586. Epub 2019 Feb 13.

Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University, Hefei, 230036, China.

Key Message: We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target. Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59-1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.
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http://dx.doi.org/10.1007/s00299-019-02387-0DOI Listing
May 2019

Comparative Transcriptome Analysis between Fertile and CMS Flower Buds in Wucai (Brassica campestris L.).

BMC Genomics 2018 Dec 12;19(1):908. Epub 2018 Dec 12.

Vegetable Genetics and Breeding Laboratory, College of Horticulture, Anhui Agricultural University, Hefei, 230036, China.

Background: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq).

Results: Microscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai.

Conclusion: The present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.
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http://dx.doi.org/10.1186/s12864-018-5331-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6292171PMC
December 2018

Shellfish/crustacean oral allergy syndrome among national service pre-enlistees in Singapore.

Asia Pac Allergy 2018 Apr 24;8(2):e18. Epub 2018 Apr 24.

Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore 308433.

Background: All Singaporean males undergo medical screening prior to compulsory military service. A history of possible food allergy may require referral to a specialist Allergy clinic to ensure that special dietary needs can be taken into account during field training and deployment.

Objective: To study the pattern of food allergy among pre-enlistees who were referred to a specialist allergy clinic to work up suspected food allergy.

Methods: Retrospective study of all pre-enlistees registered in the Clinical Immunology/Allergy New Case Registry referred to the Allergy Clinic from 1 August 2015 to 31 May 2016 for suspected food allergy.

Results: One hundred twenty pre-enlistees reporting food allergy symptoms other than rash alone were referred to the Allergy Clinic during the study period. Of these, 77 (64.2%) had food allergy. Among those with food allergy, mean age was 19.1 ± 1.5 years. They comprised predominantly Chinese (66.2%) and Malays (20.8%). The most commonly reported foods were shellfish/crustaceans (78%), peanut (15.6%), and egg (6.5%). Self-limiting oral allergy syndrome, OAS (itchy lips and throat with/without lip angioedema) was the most common manifestation (n = 33, 42.9%) followed by anaphylaxis (n = 23, 29.9%). Majority of OAS was from shellfish/crustacean (90.6%); of which shrimp (30.3%), crab (15.2%), and lobster (3.0%) were the most common. Mild childhood asthma (69.7%), allergic rhinitis (6.3%), and eczema (6.1%) were the most common atopic conditions among individuals with shellfish/crustacean OAS. This pattern was similar for shellfish/crustacean anaphylaxis. Skin prick tests were most commonly positive for shrimp (OAS 87.1% vs. anaphylaxis 100%), crab (OAS 95.8% vs. 90.9%), and lobster (OAS 91.7% vs. 63.6%).

Conclusion: OAS to shellfish/crustaceans was more common than anaphylaxis among this study population of young males referred for food allergy symptoms other than rash alone.
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http://dx.doi.org/10.5415/apallergy.2018.8.e18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931925PMC
April 2018

An in vivo microdialysis study of FLZ penetration through the blood-brain barrier in normal and 6-hydroxydopamine induced Parkinson's disease model rats.

Biomed Res Int 2014 23;2014:850493. Epub 2014 Jun 23.

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China.

FLZ (N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide) is a novel synthetic squamosamide derivative and a potential anti-Parkinson's disease (PD) agent. The objective of the present study was to investigate the penetration of free FLZ across the BBB and the effects of P-gp inhibition on FLZ transport in normal and 6-hydroxydopamine (6-OHDA) induced PD model rats. In vivo microdialysis was used to collect FLZ containing brain and blood dialysates following intravenous (i.v.) drug administration either with or without pretreatment with the specific P-gp inhibitor, zosuquidar trihydrochloride (zosuquidar·3HCl). A sensitive, rapid, and reliable ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) technique was developed and validated to quantitate free FLZ levels in the dialysates. No significant differences were observed in the brain/blood FLZ area under the concentration-time curve (AUC) ratio between normal and PD model rats. However, pretreatment with zosuquidar·3HCl markedly increased the AUC ratio in both rat models. In addition, FLZ penetration was similar in zosuquidar·3HCl-pretreated normal and PD rats. These results suggest that P-gp inhibition increases BBB permeability to FLZ, thereby supporting the hypothesis that P-gp normally restricts FLZ transfer to the brain. These findings could provide reference data for future clinical trials and may aid investigation of the BBB permeability of other CNS-active substances.
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http://dx.doi.org/10.1155/2014/850493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4090575PMC
March 2015

P-glycoprotein mediated efflux limits the transport of the novel anti-Parkinson's disease candidate drug FLZ across the physiological and PD pathological in vitro BBB models.

PLoS One 2014 18;9(7):e102442. Epub 2014 Jul 18.

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China.

FLZ, a novel anti-Parkinson's disease (PD) candidate drug, has shown poor blood-brain barrier (BBB) penetration based on the pharmacokinetic study using rat brain. P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two important transporters obstructing substrates entry into the CNS as well as in relation to PD neuropathology. However, it is unclear whether P-gp and BCRP are involved in low BBB permeability of FLZ and what the differences of FLZ brain penetration are between normal and Parkinson's conditions. For this purpose, in vitro BBB models mimicking physiological and PD pathological-related BBB properties were constructed by C6 astroglial cells co-cultured with primary normal or PD rat cerebral microvessel endothelial cells (rCMECs) and in vitro permeability experiments of FLZ were carried out. High transepithelial electrical resistance (TEER) and low permeability for sodium fluorescein (NaF) confirmed the BBB functionality of the two models. Significantly greater expressions of P-gp and BCRP were detected in PD rCMECs associated with the lower in vitro BBB permeability of FLZ in pathological BBB model compared with physiological model. In transport studies only P-gp blocker effectively inhibited the efflux of FLZ, which was consistent with the in vivo permeability data. This result was also confirmed by ATPase assays, suggesting FLZ is a substrate for P-gp but not BCRP. The present study first established in vitro BBB models reproducing PD-related changes of BBB functions in vivo and demonstrated that poor brain penetration of FLZ and low BBB permeability were due to the P-gp transport.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0102442PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4103815PMC
March 2015

Quantitative determination and pharmacokinetic study of the novel anti-Parkinson's disease candidate drug FLZ in rat brain by high performance liquid chromatography-tandem mass spectrometry.

J Pharm Biomed Anal 2012 Jul 10;66:232-9. Epub 2012 Mar 10.

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People's Republic of China.

FLZ (N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phenyl)-3-(3-methoxy-4-hydroxyphenyl)-acrylamide) is a novel anti-Parkinson's disease candidate drug. A sensitive and specific high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was developed and validated for the quantification of FLZ in rat brain. Carbamazepine was selected as the internal standard. Sample preparation involved double liquid-liquid extraction by n-hexane and ethyl acetate with high extraction efficiency. The chromatographic separation was achieved on a Zorbax SB-C(18) column (100 mm × 2.1 mm, 3.5 μm) with an isocratic elution system comprised of acetonitrile and 0.3% aqueous acetic acid at a flow rate of 0.3 ml/min. The elutes were detected under positive electrospray ionization (ESI) and the target analytes were quantified by multiple reaction monitoring (MRM) mode. The method was sensitive with the lowest limit of quantification (LLOQ) at 1.0 ng/g brain tissue. Good linearity (r>0.99) was obtained over the range of 1.0-400 ng/g. The intra- and inter-day precision ranged from 0.68% to 12%, while the accuracy between 92.7% and 111%. In addition, the stability, recovery and matrix effect involved in this method were also validated. The method was used to investigate the pharmacokinetics of FLZ in rat brain successfully after intravenous administration. The brain distribution studies showed that the brain distribution of FLZ was limited with the penetration ratio less than 0.1 in rats, with no target effect in the seven collected regions. Inhibition of P-glycoprotein (P-gp) by zosuquidar·3HCl ((2R)-1-{4-[(1aR,10bS)-1,1-difluoro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c][7]annulen-6-yl]-1-piperazinyl}-3-(5-quinolinyloxy)-2-propanol trihydrochloride) resulted in a significant increase in brain-to-plasma ratio, while no significant increase by inhibition of breast cancer resistance protein (BCRP) by ko143 (2-methyl-2-propanyl 3-[(3S,6S,12aS)-6-isobutyl-9-methoxy-1,4-dioxo-1,2,3,4,6,7,12,12a-octahydropyrazino[1',2':1,6]pyrido[3,4-b]indol-3-yl]propanoate). The results indicated that FLZ had poor penetration to the brain due to the P-gp transport system.
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http://dx.doi.org/10.1016/j.jpba.2012.03.001DOI Listing
July 2012

Simultaneous determination of seven flavonoids in dog plasma by ultra-performance liquid chromatography-tandem mass spectrometry and its application to a bioequivalence study of bioactive components in Herba Epimedii and Er-Xian Decoction.

J Pharm Biomed Anal 2011 Jan 15;54(1):186-91. Epub 2010 Jul 15.

Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, PR China.

In this study, a sensitive and specific ultra-performance liquid chromatography-tandem mass method has been developed and validated for the identification and determination of 7 flavonoids in dog plasma for the first time: epimedin A, epimedin B, epimedin C, icariin, sagittatoside B, 2″-O-rhamnosyl icariside II, and baohuoside I. Chromatographic separation was accomplished on an Agilent Zorbax-SB C(18) column (50 mm × 2.1mm, 1.8 μm) with a gradient elution system composed of 0.3% acetic acid and 0.3% acetic acid in acetonitrile at a flow rate of 0.4 mL/min. Detection was based on a triple quadrupole mass spectrometer using a multiple reaction monitoring mode with an electrospray ionization source. All of the calibration curves showed good linearity (r>0.99) within the tested concentration ranges. The lower limits of quantification of the seven analytes were all lower than 0.0654 ng/mL. The relative standard deviations for intra- and inter-batches of the seven analytes were less than 13.7% and 14.9%, respectively, at four concentration levels of quality control samples, and the recoveries were between 92.8% and 114.5%, respectively. In addition, the seven flavonoids were found to be stable in dog plasma samples under short- and long-term storage and processing conditions. The validated method was successfully applied to a bioequivalence study in dog plasma after the oral administration of extracts of Herba Epimedii and Er-Xian Decoction.
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http://dx.doi.org/10.1016/j.jpba.2010.07.010DOI Listing
January 2011
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