Publications by authors named "Zhiqian Li"

41 Publications

Aspergillus fumigatus Induces the Release of IL-8 and MCP-1 by Activating Nuclear Transcription Through Dectin-1 and CR3 Receptors in Alveolar Epithelial Cells.

Curr Microbiol 2021 Jul 16. Epub 2021 Jul 16.

Department of Hospital Infection Control and Research, Institute of Disease Control and Prevention of PLA, Beijing, China.

Invasive pulmonary aspergillosis induced by the pathogenic fungus Aspergillus fumigatus is one of the common fatal complications in immunocompromised patients. Lung epithelial cells play an important role in host immune defense against A. fumigatus. However, the interaction between lung epithelial cells and A. fumigatus conidia is not fully understood. In this study, we used the swollen conidia of A. fumigatus to stimulate the type II lung epithelial A549 cells. Results showed that swollen conidia could significantly increase RNA transcription and protein expression of interleukin 8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1), but not TNF-α in A549 cells in a time-dependent manner. Moreover, serum opsonization was able to improve the release of inflammatory factors induced by swollen conidia. Blocking of the dectin-1 or CR3 receptors, or both simultaneously, in the A549 cells could decrease the release of IL-8 and MCP-1. Additionally, blocking dectin-1 or CR3 could inhibit the transcription of nuclear factor NF-κB that was activated by swollen conidia. Here we reported for the first time that dectin-1 and CR3 receptors in A549 cells mediate the release of pro-inflammatory factors IL-8 and MCP-1 induced by A. fumigatus.
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http://dx.doi.org/10.1007/s00284-021-02534-5DOI Listing
July 2021

Amphiphilic branched polymer-nitroxides conjugate as a nanoscale agent for potential magnetic resonance imaging of multiple objects in vivo.

J Nanobiotechnology 2021 Jul 9;19(1):205. Epub 2021 Jul 9.

Huaxi MR Research Center (HMRRC), Department of Radiology, National Clinical Research Center for Geriatrics, Frontiers Science Center for Disease-Related Molecular Network, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, 610041, Chengdu, China.

Background: In order to address the potential toxicity of metal-based magnetic resonance imaging (MRI) contrast agents (CAs), a concept of non-metallic MRI CAs has emerged. Currently, paramagnetic nitroxides (such as (2,2,5,5-tetramethylpyrrolidine-1-oxyl, PROXYL), (2,2,6,6-tetramethylpiperidine-1-oxide, TEMPO), etc.) are being extensively studied because their good stability and imaging mechanism are similar to metal-based contrast agents (such as Gd chelate-based clinical CAs). However, a lower relaxivity and rapid in vivo metabolism of nitroxides remain to be addressed. Previous studies have demonstrated that the construction of macromolecular nitroxides contrast agents (mORCAs) is a promising solution through macromolecularization of nitroxides (i.e., use of large molecules to carry nitroxides). Macromolecular effects not only increase the stability of nitroxides by limiting their exposure to reductive substances in the body, but also improve the overall H water relaxation by increasing the concentration of nitroxides and slowing the molecular rotation speed.

Results: Branched pDHPMA-mPEG-Ppa-PROXYL with a high molecular weight (MW = 160 kDa) and a nitroxides content (0.059 mmol/g) can form a nanoscale (~ 28 nm) self-assembled aggregate in a water environment and hydrophobic PROXYL can be protected by a hydrophilic outer layer to obtain strong reduction resistance in vivo. Compared with a small molecular CA (3-Carboxy-PROXYL (3-CP)), Branched pDHPMA-mPEG-Ppa-PROXYL displays three prominent features: (1) its longitudinal relaxivity (0.50 mM s) is about three times that of 3-CP (0.17 mM s); (2) the blood retention time of nitroxides is significantly increased from a few minutes of 3-CP to 6 h; (3) it provides long-term and significant enhancement in MR imaging of the tumor, liver, kidney and cardiovascular system (heart and aortaventralis), and this is the first report on nitroxides-based MRI CAs for imaging the cardiovascular system.

Conclusions: As a safe and efficient candidate metal-free magnetic resonance contrast agent, Branched pDHPMA-mPEG-Ppa-PROXYL is expected to be used not only in imaging the tumor, liver and kidney, but also the cardiovascular system, which expands the application scope of these CAs.
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http://dx.doi.org/10.1186/s12951-021-00951-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272293PMC
July 2021

Dendronized hyaluronic acid-docetaxel conjugate as a stimuli-responsive nano-agent for breast cancer therapy.

Carbohydr Polym 2021 Sep 7;267:118160. Epub 2021 May 7.

Huaxi MR Research Center (HMRRC), Department of Radiology, National Clinical Research Center for Geriatrics, Department of Breast Surgery, Clinical Research Center for Breast, Functional and Molecular Imaging Key Laboratory of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, China. Electronic address:

To achieve target delivery of anti-tumor drugs with great biocompatibility into tumor tissues, a stimuli-responsive dendronized hyaluronic acid (HA)-docetaxel conjugate (HA-DTX-Dendron, HADD) was designed and prepared. The incorporation of HA in HADD improved the delivery of DTX to tumor cells with rich CD44 receptors. Enhanced biocompatibility and therapeutic outcomes were achieved using glyodendrons-modified HA and tumor microenvironment-responsive linkers in HADD. The glycodendron was connected with HA via GSH-responsive disulfide bonds, and the drug DTX was linked to the carrier via a cathepsin B-responsive tetrapeptide GFLG. This design resulted in self-assembly nanostructures for facilitating uptake of HADD by tumor cells and rapid release of DTX to exert its therapeutic effect. Compared to free DTX, HADD showed much higher tumor growth inhibition in the MDA-MB-231 tumor-bearing mice model (up to 99.71%), and no toxicity was observed. Therefore, HADD could be employed as an efficacious nano-agent for treating triple negative breast cancer (TNBC).
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http://dx.doi.org/10.1016/j.carbpol.2021.118160DOI Listing
September 2021

Two dehydroecdysone reductases act as fat body-specific 20E catalyzers in Bombyx mori.

Insect Sci 2021 May 20. Epub 2021 May 20.

Jiangsu Key Laboratory of Sericultural Biology and Biotechnology, School of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu Province, 212100, China.

Periodic post-embryonic changes in insects, including growth, development and metamorphosis, are strictly controlled by many compounds, including steroid hormones. The biosynthesis and clearance of 20-hydroxyecdysone (20E), the major active form of the insect steroid hormone ecdysone, result in titer fluctuations that help control insect development. The inactivation of 20E in the silkworm Bombyx mori is highly tissue-specific, with CYP18A1 and ecdysone oxidase controlling 20E inactivation specifically in the mid-silk gland and midgut, respectively. Here, we characterized silkworm 3-dehydroecdysone 3α reductase (Bm3DE3α) and 3-dehydroecdysone 3β reductase (Bm3DE3β), two enzymes involved predominantly in the C-3-mediated catalysis of 20E in fat bodies. The ubiquitous and silk gland-specific overexpression of Bm3DE3α decreased the 20E titer, resulting in larval lethality and larval-pupal transition failure, respectively. In contrast, the ubiquitous and mid-silk gland-specific overexpression of Bm3DE3β increased the 20E titer, resulting in larval growth delays and lethality at the mid-fifth larval stage, respectively. Thus, Bm3DE3α and Bm3DE3β mediate fat body-specific steroid hormone metabolism in B. mori, indicating that highly diversified 20E metabolism-related mechanisms exist in different insect species.
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http://dx.doi.org/10.1111/1744-7917.12928DOI Listing
May 2021

CopyCatchers are versatile active genetic elements that detect and quantify inter-homolog somatic gene conversion.

Nat Commun 2021 05 11;12(1):2625. Epub 2021 May 11.

Section of Cell and Developmental Biology, University of California San Diego, La Jolla, CA, USA.

CRISPR-based active genetic elements, or gene-drives, copied via homology-directed repair (HDR) in the germline, are transmitted to progeny at super-Mendelian frequencies. Active genetic elements also can generate widespread somatic mutations, but the genetic basis for such phenotypes remains uncertain. It is generally assumed that such somatic mutations are generated by non-homologous end-joining (NHEJ), the predominant double stranded break repair pathway active in somatic cells. Here, we develop CopyCatcher systems in Drosophila to detect and quantify somatic gene conversion (SGC) events. CopyCatchers inserted into two independent genetic loci reveal unexpectedly high rates of SGC in the Drosophila eye and thoracic epidermis. Focused RNAi-based genetic screens identify several unanticipated loci altering SGC efficiency, one of which (c-MYC), when downregulated, promotes SGC mediated by both plasmid and homologous chromosome-templates in human HEK293T cells. Collectively, these studies suggest that CopyCatchers can serve as effective discovery platforms to inform potential gene therapy strategies.
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http://dx.doi.org/10.1038/s41467-021-22927-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113449PMC
May 2021

VvHDZ28 positively regulate salicylic acid biosynthesis during seed abortion in Thompson Seedless.

Plant Biotechnol J 2021 Apr 9. Epub 2021 Apr 9.

College of Horticulture, Northwest A&F University, Yangling, China.

Seedlessness in grapes is one of the features most appreciated by consumers. However, the mechanisms underlying seedlessness in grapes remain obscure. Here, we observe small globular embryos and globular embryos in Pinot Noir and Thompson Seedless from 20 to 30 days after flowering (DAF). From 40 to 50 DAF, we observe torpedo embryos and cotyledon embryos in Pinot Noir but aborted embryos and endosperm in Thompson Seedless. Thus, RNA-Seq analyses of seeds at these stages from Thompson Seedless and Pinot Noir were performed. A total of 6442 differentially expressed genes were identified. Among these, genes involved in SA biosynthesis, VvEDS1 and VvSARD1, were more highly expressed in Thompson Seedless than in Pinot Noir. Moreover, the content of endogenous SA is at least five times higher in Thompson Seedless than in Pinot Noir. Increased trimethylation of H3K27 of VvEDS1 and VvSARD1 may be correlated with lower SA content in Pinot Noir. We also demonstrate that VvHDZ28 positively regulates the expression of VvEDS1. Moreover, over-expression of VvHDZ28 results in seedless fruit and increased SA contents in Solanum lycopersicum. Our results reveal the potential role of SA and feedback regulation of VvHDZ28 in seedless grapes.
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http://dx.doi.org/10.1111/pbi.13596DOI Listing
April 2021

Genome-wide identification of HSF family in peach and functional analysis of involvement in root and aerial organ development.

PeerJ 2021 12;9:e10961. Epub 2021 Mar 12.

College of Horticulture, Henan Agricultural University, Zhengzhou, China.

Background: Heat shock factors (HSFs) play important roles during normal plant growth and development and when plants respond to diverse stressors. Although most studies have focused on the involvement of HSFs in the response to abiotic stresses, especially in model plants, there is little research on their participation in plant growth and development or on the HSF (PpHSF) gene family in peach ().

Methods: DBD (PF00447), the HSF characteristic domain, was used to search the peach genome and identify . Phylogenetic, multiple alignment and motif analyses were conducted using MEGA 6.0, ClustalW and MEME, respectively. The function of was confirmed by overexpression of into Arabidopsis.

Results: Eighteen genes were identified within the peach genome. The genes were nonuniformly distributed on the peach chromosomes. Seventeen of the (94.4%) contained one or two introns, except , which contained three introns. The in silico-translated PpHSFs were classified into three classes (PpHSFA, PpHSFB and PpHSFC) based on multiple alignment, motif analysis and phylogenetic comparison with HSFs from and . Dispersed gene duplication (DSD at 67%) mainly contributed to HSF gene family expansion in peach. Promoter analysis showed that the most common cis-elements were the MYB (abiotic stress response), ABRE (ABA-responsive) and MYC (dehydration-responsive) elements. Transcript profiling of 18 showed that the expression trend of was consistent with shoot length changes in the cultivar 'Zhongyoutao 14'. Further analysis of the was conducted in 5-year-old peach trees, and , respectively. Tissue-specific expression analysis showed that was expressed predominantly in young vegetative organs (leaf and apex). Subcellular localization revealed that PpHSF5 was located in the nucleus in cells. Two transgenic Arabidopsis lines were obtained that overexpressed . The root length and the number of lateral roots in the transgenic seedlings were significantly less than in WT seedlings and after cultivation for three weeks. The transgenic rosettes were smaller than those of the WT at 2-3 weeks. The two transgenic lines exhibited a dwarf phenotype three weeks after transplanting, although there was no significant difference in the number of internodes. Moreover, the PpHSF5-OE lines exhibited enhanced thermotolerance. These results indicated that PpHSF5 might be act as a suppresser of growth and development of root and aerial organs.
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http://dx.doi.org/10.7717/peerj.10961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958895PMC
March 2021

Dendron-Functionalized Polyglutamate-Pyropheophorbide-a Conjugates as Nanomedicines for Breast Cancer Photodynamic Therapy.

Macromol Rapid Commun 2021 May 24;42(9):e2100013. Epub 2021 Mar 24.

Huaxi MR Research Center (HMRRC), Department of Radiology, Functional and Molecular Imaging Key Laboratory of Sichuan Province, West China Hospital, Sichuan University, Chengdu, 610041, China.

Photodynamic therapy (PDT) has shown its promise in the treatment of cancer. Herein, a dendron-functionalized polyglutamic acid (PGA) polymer (PG-L8G-Ppa-Dendron, PGPD) is synthesized and it is conjugated with pyropheophorbide-a (Ppa) for the first time to treat triple negative breast cancer (TNBC), whereas a linear polyglutamate-Ppa conjugate (PGP) is synthesized as a control. Compared to the linear counterpart, the glycosylated polymer-based PGPD with a dendritic structure has excellent solubility and it self-assembles to form uniform-sized nanoparticles. PGPD displays a highly effective PDT effect in the animal model, evidenced with effective induction of reactive oxygen species (ROS) production and cell apoptosis. This may be due to an enhanced efficiency in delivery and accumulation of Ppa by this glycosylated dendritic polymer at tumor sites. Therefore, PGPD can be a highly effective and biosafe nanoagent for PDT of TNBC.
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http://dx.doi.org/10.1002/marc.202100013DOI Listing
May 2021

Functional Analysis of the Gene Family in Peach.

Front Plant Sci 2021 17;12:619158. Epub 2021 Feb 17.

College of Horticulture, Henan Agricultural University, Zhengzhou, China.

Peach ( L. Batsch) trees grow vigorously and are subject to intense pruning during orchard cultivation. Reducing the levels of endogenous gibberellins (GAs) represents an effective method for controlling branch growth. Gibberellin 2-oxidases (GA2oxs) deactivate bioactive GAs, but little is known about the GA2ox gene family in peach. In this study, we identified seven genes in the peach genome, which were clustered into three subgroups: C-GA2ox-I, C-GA2ox-II, and C-GA2ox-I. Overexpressing representative genes from the three subgroups, , , and , in tobacco resulted in dwarf plants with shorter stems and smaller leaves than the wild type. An analysis of the GA metabolic profiles of the transgenic plants showed that PpGA2ox-5 (a member of subgroup C-GA2ox-II) is simultaneously active against both C-GAs and C-GAs,which implied that C-GA2ox-II enzymes represent intermediates of C-GA2oxs and C-GA2oxs. Exogenous GA treatment of shoot tips activated the expression of all seven genes, with different response times: the - genes were transcriptionally activated more rapidly than the genes. GA metabolic profile analysis suggested that C-GA2ox depletes GA levels more broadly than C-GA2ox. These results suggest that the gene family is responsible for fine-tuning endogenous GA levels in peach. Our findings provide a theoretical basis for appropriately controlling the vigorous growth of peach trees.
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http://dx.doi.org/10.3389/fpls.2021.619158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928363PMC
February 2021

Cathepsin B-responsive and gadolinium-labeled branched glycopolymer-PTX conjugate-derived nanotheranostics for cancer treatment.

Acta Pharm Sin B 2021 Feb 14;11(2):544-559. Epub 2020 Aug 14.

Huaxi MR Research Center (HMRRC), Department of Neurosurgery, and Department of Radiology, Functional and Molecular Imaging Key Laboratory of Sichuan Province, West China Hospital, Sichuan University, Chengdu 610041, China.

Multi-modal therapeutics are emerging for simultaneous diagnosis and treatment of cancer. Polymeric carriers are often employed for loading multiple drugs due to their versatility and controlled release of these drugs in response to a tumor specific microenvironment. A theranostic nanomedicine was designed and prepared by complexing a small gadolinium chelate, conjugating a chemotherapeutic drug PTX through a cathepsin B-responsive linker and covalently bonding a fluorescent probe pheophorbide a (Ppa) with a branched glycopolymer. The branched prodrug-based nanosystem was degradable in the tumor microenvironment with overexpressed cathepsin B, and PTX was simultaneously released to exert its therapeutic effect. The theranostic nanomedicine, branched glycopolymer-PTX-DOTA-Gd, had an extended circulation time, enhanced accumulation in tumors, and excellent biocompatibility with significantly reduced gadolinium ion (Gd) retention after 96 h post-injection. Enhanced imaging contrast up to 24 h post-injection and excellent antitumor efficacy with a tumor inhibition rate more than 90% were achieved from glycopolymer-PTX-DOTA-Gd without obvious systematic toxicity. This branched polymeric prodrug-based nanomedicine is very promising for safe and effective diagnosis and treatment of cancer.
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http://dx.doi.org/10.1016/j.apsb.2020.07.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893117PMC
February 2021

Tumor microenvironment-responsive PEGylated heparin-pyropheophorbide-a nanoconjugates for photodynamic therapy.

Carbohydr Polym 2021 Mar 13;255:117490. Epub 2020 Dec 13.

Huaxi MR Research Center (HMRRC), Department of Radiology, West China Hospital, Functional and Molecular Imaging Key Laboratory of Sichuan Province, Sichuan University, Chengdu, 610041, China. Electronic address:

To deliver photosensitizers with PEGylated heparin (HP) into tumor cells for photodynamic therapy, we prepared two polyethylene glycol (PEG)-functionalized HP-based polymers conjugated with pyropheophorbide-a (Ppa): a non-GSH-responsive nanoagent (HP-Ppa-mPEG) with the mPEG moiety chemically attached to HP directly; and a GSH-responsive nanoagent (HP-Ppa-SS-mPEG) with the mPEG moiety conjugated to HP via a disulfide linkage. The Ppa-functionalized HP without PEGylation (HP-Ppa) was designed as another control. These amphiphilic polymers could aggregate into nanoparticles. Cellular uptake of three nanoparticles by 4T1 cells led to abundant production of reactive oxygen species after irradiation by a 660 nm laser, inducing cell apoptosis. HP-Ppa-SS-mPEG was found to achieve the highest tumor accumulation, the longest retention time and the best penetration into tumor tissues, resulting in the highest in vivo anticancer efficacy with 94.3 % tumor growth inhibition rate, suggesting that tumor microenvironment-responsive PEGylated HP-based nanomedicines may act as efficient anticancer agents.
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http://dx.doi.org/10.1016/j.carbpol.2020.117490DOI Listing
March 2021

Assessment of White Matter Lesions in Parkinson's Disease: Voxel-based Analysis and Tract-based Spatial Statistics Analysis of Parkinson's Disease with Mild Cognitive Impairment.

Curr Neurovasc Res 2020 ;17(4):480-486

Department of Radiology, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou 550004, China.

Objective: Application of diffusion tensor imaging (DTI) to explore the changes of FA value in patients with Parkinson's disease (PD) with mild cognitive impairment.

Methods: 27 patients with PD were divided into PD with mild cognitive impairment (PD-MCI) group (n = 7) and PD group (n = 20). The original images were processed using voxel-based analysis (VBA) and tract-based spatial statistics (TBSS).

Results: The average age of pd-mci group was longer than that of PD group, and the course of disease was longer than that of PD group. Compared with PD group, the voxel based analysis-fractional anisotropy (VBA-FA) values of PD-MCI group decreased in the following areas: bilateral frontal lobe, bilateral temporal lobe, bilateral parietal lobe, bilateral subthalamic nucleus, corpus callosum, and gyrus cingula. Tract-based spatial statistics-fractional anisotropy (TBSS-FA) values in PD-MCI group decreased in bilateral corticospinal tract, anterior cingulum, posterior cingulum, fornix tract, bilateral superior thalamic radiation, corpus callosum(genu, body and splenium), bilateral uncinate fasciculus, bilateral inferior longitudinal fasciculus, bilateral superior longitudinal fasciculus, bilateral superior fronto-occipital fasciculus, bilateral inferior fronto-occipital fasciculus, and bilateral parietal-occipital tracts. The mean age of onset in the PD-MCI group was greater than that in the PD group, and the disease course was longer than that in the PD group.

Conclusion: DTI-based VBA and TBSS post-processing methods can detect abnormalities in multiple brain areas and white matter fiber tracts in PD-MCI patients. Impairment of multiple cerebral cortex and white matter fiber pathways may be an important causes of cognitive dysfunction in PD-MCI.
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http://dx.doi.org/10.2174/1567202617666200901181842DOI Listing
January 2020

Glycodendron/pyropheophorbide-a (Ppa)-functionalized hyaluronic acid as a nanosystem for tumor photodynamic therapy.

Carbohydr Polym 2020 Nov 12;247:116749. Epub 2020 Jul 12.

Huaxi MR Research Center (HMRRC), Department of Radiology, Functional and Molecular Imaging Key Laboratory of Sichuan Province, National Clinical Research Center for Geriatrics, and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China. Electronic address:

To enhance the drug delivery efficiency of hyaluronic acid (HA), we designed and prepared glycodendron and pyropheophorbide-a (Ppa)-functionalized HA (HA-Ppa-Dendron) as a nanosystem for cancer photodynamic therapy. Linear Ppa-modified HA (HA-Ppa) was also prepared as a control. Cellular uptake of both polymers by MDA-MB-231 cells led to mitochondrial dysfunction and generation of reactive oxygen species under the irradiation of a laser. Compared to the linear polymer, HA-Ppa-Dendron had higher molecular weight, a more compact nanoscale particle size, and a dendritic structure, resulting in a much longer blood circulation time and higher tumor accumulation. HA-Ppa-Dendron outperformed HA-Ppa in inhibiting cell growth, with 60 % of tumors was eradicated under laser irradiation. Tumor growth inhibition (TGI) up to 99.2 % was achieved from HA-Ppa-Dendron, which was much higher than that of HA-Ppa (50.6 %). Therefore, glycodendron-functionalized HAs by integration of HA and dendritic polymers may act as efficient anti-cancer nanomedicine.
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http://dx.doi.org/10.1016/j.carbpol.2020.116749DOI Listing
November 2020

Enhancing the Efficacy of Metal-Free MRI Contrast Agents via Conjugating Nitroxides onto PEGylated Cross-Linked Poly(Carboxylate Ester).

Adv Sci (Weinh) 2020 Jul 3;7(14):2000467. Epub 2020 Jun 3.

Huaxi MR Research Center (HMRRC), Department of Radiology, Functional and molecular imaging Key Laboratory of Sichuan Province, National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital Sichuan University Chengdu 610041 P. R. China.

Herein, two water-soluble PROXYL-based magnetic resonance imaging (MRI) macromolecular organic contrast agents (mORCAs) are designed and synthesized: linear and cross-linked PCE-mPEG-Ppa-PROXYL. They are prepared by conjugating linear and cross-linked poly(carboxylate ester) (PCE) with poly(ethylene glycol) (mPEG)-modified nitroxides (PROXYL), respectively. Both mORCAs form self-assembled aggregates in an aqueous phase and PROXYL is protected inside a hydrophobic core to achieve great resistance to reduction in the physiological environment, and they have low toxicity. Since cross-linked PCE-mPEG-Ppa-PROXYL possess a branched architecture, its self-assembled aggregate is more stable and compact with a greater particle size. Cross-linked PCE-mPEG-Ppa-PROXYL outperform the linear one in the following aspects: 1) its longitudinal relaxivity (  = 0.79 mm s) is higher than that of the linear one (  = 0.64 mm s) and both excel the best mORCA reported so far (  = 0.42 mm s); 2) its blood retention time (≈48 h) is longer than that of its linear counterpart (≈10 h); 3) cross-linked PCE-mPEG-Ppa-PROXYL provided better MR imaging contrast resolution in normal organs (liver and kidney) and tumor of mice than the linear one. Overall, cross-linked PCE-mPEG-Ppa-PROXYL may have great potential to be a novel metal-free macromolecular contrast agent for MR imaging.
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http://dx.doi.org/10.1002/advs.202000467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7375229PMC
July 2020

A Nanostrategy for Efficient Imaging-Guided Antitumor Therapy through a Stimuli-Responsive Branched Polymeric Prodrug.

Adv Sci (Weinh) 2020 Mar 31;7(6):1903243. Epub 2020 Jan 31.

Huaxi MR Research Center (HMRRC) Department of Radiology Functional and Molecular Imaging Key Laboratory of Sichuan Province National Clinical Research Center for Geriatrics State Key Laboratory of Biotherapy West China Hospital Sichuan University Chengdu 610041 China.

A stimuli-responsive polymeric prodrug-based nanotheranostic system with imaging agents (cyanine5.5 and gadolinium-chelates) and a therapeutic agent paclitaxel (PTX) is prepared via polymerization and conjugating chemistry. The branched polymeric PTX-Gd-based nanoparticles (BP-PTX-Gd NPs) demonstrate excellent biocompatibility, and high stability under physiological conditions, but they stimuli-responsively degrade and release PTX rapidly in a tumor microenvironment. The in vitro behavior of NPs labeled with fluorescent dyes is effectively monitored, and the NPs display high cytotoxicity to 4T1 cells similar to free PTX by impairing the function of microtubules, downregulating anti-apoptotic protein Bcl-2, and upregulating the expression of Bax, cleaved caspase-3, cleaved caspase-9, cleaved-PARP, and p53 proteins. Great improvement in magnetic resonance imaging (MRI) is demonstrated by these NPs, and MRI accurately maps the temporal change profile of the tumor volume after injection of NPs and the tumor treatment process is also closely correlated with the values measured from MRI, demonstrating the capability of providing real-time feedback to the chemotherapeutic treatment effectiveness. The imaging-guided chemotherapy to the 4T1 tumor in the mice model achieves an excellent anti-tumor effect. This stimuli-responsive polymeric nano-agent opens a new door for efficient breast cancer treatment under the guidance of fluorescence/MRI.
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http://dx.doi.org/10.1002/advs.201903243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080516PMC
March 2020

Dendronized-Polymer Disturbing Cells' Stress Protection by Targeting Metabolism Leads to Tumor Vulnerability.

Adv Mater 2020 Apr 21;32(14):e1907490. Epub 2020 Feb 21.

Huaxi MR Research Center (HMRRC), Department of Radiology, Functional and molecular imaging Key Laboratory of Sichuan Province, National Clinical Research Center for Geriatrics, State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, 610041, China.

Metabolic demand of cancer is quite unique compared to normal tissues and this is an emerging hallmark of cancer, which brings a potential opportunity to discover drugs that target cancer cell metabolism. Herein, the development of a dendronized pyropheophorbide a (Ppa)-conjugated polymer (DPP) is reported, and a linear Ppa-conjugated polymer (LPP) is reported as a control. DPP is found to disturb cellular metabolism including increased energy depletion, dysfunctional H regulation, and decreased antioxidation, resulting in deficiency in protecting cells from stresses. These vulnerable cells are subjected to photodynamic therapy (PDT) treatment in the presence of DPP, resulting in attenuated cancer cell growth and eventually cell death. The in vivo anticancer efficacy is also ascribed to significantly prolonged blood circulation and enhanced tumor accumulation of DPP due to its unique molecular structure. This study presents a new platform using dendronized polymers for tumor suppression by targeting cancer cell metabolism.
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http://dx.doi.org/10.1002/adma.201907490DOI Listing
April 2020

Genome-wide identification and transcriptome profiling reveal that E3 ubiquitin ligase genes relevant to ethylene, auxin and abscisic acid are differentially expressed in the fruits of melting flesh and stony hard peach varieties.

BMC Genomics 2019 Nov 21;20(1):892. Epub 2019 Nov 21.

College of Horticulture, Henan Agricultural University, Zhengzhou, 450002, China.

Background: Ubiquitin ligases (E3) are the enzymes in the ubiquitin/26S proteasome pathway responsible for targeting proteins to the degradation pathway and play major roles in multiple biological activities. However, the E3 family and their functions are yet to be identified in the fruit of peach.

Results: In this study, genome-wide identification, classification and characterization of the E3 ligase genes within the genome of peach (Prunus persica) was carried out. In total, 765 E3 (PpE3) ligase genes were identified in the peach genome. The PpE3 ligase genes were divided into eight subfamilies according to the presence of known functional domains. The RBX subfamily was not detected in peach. The PpE3 ligase genes were not randomly distributed among the 8 chromosomes, with a greater concentration on the longer chromosomes. The primary mode of gene duplication of the PpE3 ligase genes was dispersed gene duplication (DSD). Four subgroups of the BTB subfamily never characterized before were newly identified in peach, namely BTBAND, BTBBL, BTBP and BTBAN. The expression patterns of the identified E3 ligase genes in two peach varieties that display different types of fruit softening (melting flesh, MF, and stony hard, SH) were analyzed at 4 different stages of ripening using Illumina technology. Among the 765 PpE3 ligase genes, 515 (67.3%) were expressed (FPKM > 1) in the fruit of either MF or SH during fruit ripening. In same-stage comparisons, 231 differentially expressed genes (DEGs) were identified between the two peach cultivars. The number of DEGs in each subfamily varied. Most DEGs were members of the BTB, F-box, U-box and RING subfamilies. PpE3 ligase genes predicted to be involved in ethylene, auxin, or ABA synthesis or signaling and DNA methylation were differentially regulated. Eight PpE3 ligase genes with possible roles in peach flesh texture and fruit ripening were discussed.

Conclusions: The results of this study provide useful information for further understanding the functional roles of the ubiquitin ligase genes in peach. The findings also provide the first clues that E3 ligase genes may function in the regulation of peach ripening.
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http://dx.doi.org/10.1186/s12864-019-6258-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873611PMC
November 2019

Transcriptomic analysis of Chinese wild Vitis pseudoreticulata in response to Plasmopara viticola.

Protoplasma 2019 Sep 21;256(5):1409-1424. Epub 2019 May 21.

State Key Laboratory of Crop Stress Biology for Arid Areas, Northwest A&F University, Yangling, Shaanxi, People's Republic of China.

Downy mildew, resulted from Plasmopara viticola, is one of most severe fungal diseases of grapevine. Since Vitis vinifera is susceptible to downy mildew, much effort has been focused on improving the resistance of V. vinifera. The Chinese wild V. pseudoreticulata accession Baihe-35-1 (BH) shows resistance to P. viticola; however, the molecular mechanism underlying its resistance to P. viticola is largely unknown. In order to better understand the cellular processes, the transcriptomic changes were investigated at 0, 12, 24, 48, 96, and 120 h post infection (hpi). Transcriptome analysis identified a total of 175 differentially expressed genes. Most of them were found to be associated with oxidative stress, cell wall modification, and protein modification. Moreover, the BH resistance to P. viticola was involved in metabolism process, including terpene synthesis and hormone synthesis. In addition, we verified 12 genes to ensure the accuracy of transcriptome data using quantitative real-time PCR (qRT-PCR). This study broadly characterizes a molecular mechanism in which oxidative stress and cell wall biosynthesis and modification play important roles in the response of BH to P. viticola and provides a basis for further analysis of key genes involved in the resistance to P. viticola.
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http://dx.doi.org/10.1007/s00709-019-01387-xDOI Listing
September 2019

Genome-wide analysis of glyoxalase-like gene families in grape (Vitis vinifera L.) and their expression profiling in response to downy mildew infection.

BMC Genomics 2019 May 9;20(1):362. Epub 2019 May 9.

College of Horticulture, Northwest A&F University, Yangling, Shaanxi, People's Republic of China.

Background: The glyoxalase system usually comprises two enzymes, glyoxalase I (GLYI) and glyoxalase II (GLYII). This system converts cytotoxic methylglyoxal (MG) into non-toxic D-lactate in the presence of reduced glutathione (GSH) in two enzymatic steps. Recently, a novel type of glyoxalase III (GLYIII) activity has observed in Escherichia coli that can detoxify MG into D-lactate directly, in one step, without a cofactor. Investigation of the glyoxalase enzymes of a number of plant species shows the importance of their roles in response both to abiotic and to biotic stresses. Until now, glyoxalase gene families have been identified in the genomes of four plants, Arabidopsis, Oryza sativa, Glycine max and Medicago truncatula but no similar study has been done with the grapevine Vitis vinifera L.

Results: In this study, four GLYI-like, two GLYII-like and three GLYIII-like genes are identified from the genome database of grape. All these genes were analysed in detail, including their chromosomal locations, phylogenetic relationships, exon-intron distributions, protein domain organisations and the presence of conserved binding sites. Using quantitative real-time PCR analysis (qRT-PCR), the expression profiles of these genes were analysed in different tissues of grape, and also when under infection stress from downy mildew (Plasmopara viticola). The study reveals that most VvGLY-like genes had higher expressions in stem, leaf, tendril and ovule but lower expressions in the flower. In addition, most of the VvGLY-like gene members were P. viticola responsive with high expressions 6-12 h and 96-120 h after inoculation. However, VvGLYI-like1 was highly expressed 48 h after inoculation, similar to VvPR1 and VvNPR1 which are involved in the defence response.

Conclusions: This study identified the GLYI-like, GLYII-like and GLYIII-like full gene families of the grapevine. Based on a phylogenetic analysis and the presence of conserved binding sites, we speculate that these glyoxalase-like genes in grape encode active glyoxalases. Moreover, our study provides a basis for discussing the roles of VvGLYI-like, VvGLYII-like and VvGLYIII-like genes in grape's response to downy mildew infection. Our results shed light on the selection of candidate genes for downy mildew tolerance in grape and lay the foundation for further functional investigations of these glyoxalase genes.
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http://dx.doi.org/10.1186/s12864-019-5733-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509763PMC
May 2019

Gliotoxin destructs the pulmonary epithelium barrier function by reducing cofilin oligomer formation to promote the dissolution of actin stress fibers.

Microb Pathog 2018 Oct 11;123:169-176. Epub 2018 Jul 11.

Institute for Disease Control and Prevention of PLA, Beijing, China. Electronic address:

The destruction of pulmonary epithelium is a major feature of lung diseases caused by the fungal pathogen Aspergillus fumigatus (A. fumigatus). Gliotoxin, a major mycotoxin of A. fumigatus, is widely postulated to be associated with the tissue invasion. However, the mechanism is unclear. In this study, we first discovered that cofilin, a regulator of actin dynamics in the pulmonary epithelial cells, existed mainly in the form of oligomer, which kept it unable to depolymerize actin filaments. Gliotoxin could reduce the formation of cofilin oligomer and promote the release of active cofilin monomer by regulating cofilin phosphorylation balance. Then, the active cofilin induced the dissolution of actin stress fibers to result in the disruption of pulmonary epithelium barrier function. Collectively, our study revealed a novel mechanism of gliotoxin destructing lung epithelium barrier function and for the first time indicated the role of cofilin oligomer in this process.
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http://dx.doi.org/10.1016/j.micpath.2018.07.007DOI Listing
October 2018

Bombyx mori histone methyltransferase BmAsh2 is essential for silkworm piRNA-mediated sex determination.

PLoS Genet 2018 02 23;14(2):e1007245. Epub 2018 Feb 23.

CAS Key Laboratory of Insect Developmental and Evolutionary Biology, CAS Center for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China.

Sex determination is a hierarchically-regulated process with high diversity in different organisms including insects. The W chromosome-derived Fem piRNA has been identified as the primary sex determination factor in the lepidopteran insect, Bombyx mori, revealing a distinctive piRNA-mediated sex determination pathway. However, the comprehensive mechanism of silkworm sex determination is still poorly understood. We show here that the silkworm PIWI protein BmSiwi, but not BmAgo3, is essential for silkworm sex determination. CRISPR/Cas9-mediated depletion of BmSiwi results in developmental arrest in oogenesis and partial female sexual reversal, while BmAgo3 depletion only affects oogenesis. We identify three histone methyltransferases (HMTs) that are significantly down-regulated in BmSiwi mutant moths. Disruption one of these, BmAsh2, causes dysregulation of piRNAs and transposable elements (TEs), supporting a role for it in the piRNA signaling pathway. More importantly, we find that BmAsh2 mutagenesis results in oogenesis arrest and partial female-to-male sexual reversal as well as dysregulation of the sex determination genes, Bmdsx and BmMasc. Mutagenesis of other two HMTs, BmSETD2 and BmEggless, does not affect piRNA-mediated sex determination. Histological analysis and immunoprecipitation results support a functional interaction between the BmAsh2 and BmSiwi proteins. Our data provide the first evidence that the HMT, BmAsh2, plays key roles in silkworm piRNA-mediated sex determination.
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http://dx.doi.org/10.1371/journal.pgen.1007245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841826PMC
February 2018

Grapevine VpPR10.1 functions in resistance to Plasmopara viticola through triggering a cell death-like defence response by interacting with VpVDAC3.

Plant Biotechnol J 2018 08 8;16(8):1488-1501. Epub 2018 Mar 8.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F University, Yangling, Shaanxi, China.

As one of the most serious diseases in grape, downy mildew caused by Plasmopara viticola is a worldwide grape disease. Much effort has been focused on improving susceptible grapevine resistance, and wild resistant grapevine species are important for germplasm improvement of commercial cultivars. Using yeast two-hybrid screen followed by a series of immunoprecipitation experiments, we identified voltage-dependent anion channel 3 (VDAC3) protein from Vitis piasezkii 'Liuba-8' as an interacting partner of VpPR10.1 cloned from Vitis pseudoreticulata 'Baihe-35-1', which is an important germplasm for its resistance to a range of pathogens. Co-expression of VpPR10.1/VpVDAC3 induced cell death in Nicotiana benthamiana, which accompanied by ROS accumulation. VpPR10.1 transgenic grapevine line showed resistance to P. viticola. We conclude that the VpPR10.1/VpVDAC3 complex is responsible for cell death-mediated defence response to P. viticola in grapevine.
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http://dx.doi.org/10.1111/pbi.12891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6041444PMC
August 2018

Evolution and expression analysis reveal the potential role of the HD-Zip gene family in regulation of embryo abortion in grapes (Vitis vinifera L.).

BMC Genomics 2017 Sep 21;18(1):744. Epub 2017 Sep 21.

College of Horticulture, Northwest A&F University, Yangling, Shaanxi, People's Republic of China.

Background: The HD-Zip family has a diversity of functions during plant development. In this study, we identify 33 HD-Zip transcription factors in grape and detect their expressions in ovules and somatic embryos, as well as in various vegetative organs.

Results: A genome-wide survey for HD-Zip transcription factors in Vitis was conducted based on the 12 X grape genome (V. vinifera L.). A total of 33 members were identified and classified into four subfamilies (I-IV) based on phylogeny analysis with Arabidopsis, rice and maize. VvHDZs in the same subfamily have similar protein motifs and intron/exon structures. An evaluation of duplication events suggests several HD-Zip genes arose before the divergence of the grape and Arabidopsis lineages. The 33 members of HD-Zip were differentially expressed in ovules of the stenospermic grape, Thompson Seedless and of the seeded grape, Pinot noir. Most have higher expressions during ovule abortion in Thompson Seedless. In addition, transcripts of the HD-Zip family were also detected in somatic embryogenesis of Thompson Seedless and in different vegetative organs of Thompson Seedless at varying levels. Additionally, VvHDZ28 is located in the nucleus and had transcriptional activity consistent with the typical features of the HD-Zip family. Our results provide a foundation for future grape HD-Zip gene function research.

Conclusions: The identification and expression profiles of the HD-Zip transcription factors in grape, reveal their diverse roles during ovule abortion and organ development. Our results lay a foundation for functional analysis of grape HDZ genes.
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http://dx.doi.org/10.1186/s12864-017-4110-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5609062PMC
September 2017

A pilot study of pre-operative motor dysfunction from gliomas in the region of corticospinal tract: Evaluation with diffusion tensor imaging.

PLoS One 2017 22;12(8):e0182795. Epub 2017 Aug 22.

Department of Radiology, Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, People's Republic of China.

Background And Purpose: Brain tumors in the corticospinal tract (CST) region are more likely to cause motor dysfunction. The aim of this study is to evaluate the effect of gliomas located in the CST region on motor function with diffusion tensor imaging (DTI) preoperatively.

Materials And Methods: Forty-five patients with histopathologically confirmed gliomas were included in this pilot study, in all cases (low-grade n = 13, high-grade n = 32) CST but not the motor cortex were involved by the tumor. DTI image were acquired and the posterior limb of the internal capsule fractional anisotropy (FA) and relative FA (rFA = affected FA/contralateral FA) were measured. Injury of the CST from tumor was divided into three grades (grade 1: displacement, grade 2: displacement and infiltration, grade 3: displacement and disruption). The fiber density index (FDi) and relative FDi (rFDi = affected FDi/contralateral FDi) of the injured and contralateral CST were measured. The correlations between muscle strength and the CST injury grade and the rFA, affected FDi, rFDi values were calculated using Spearman rank correlation analysis. rFA and rFDi values of muscle strength groups (MMT2-5) were compared with one-way analysis of variance (ANOVA). The difference of muscle strength between low- and high-grade glioma groups were analysed with the Mann-Whitney U-test.

Results: Muscle strength was negatively correlated with the injury grade of the CST (r = -0.840, P<0.001). Muscle strength was positively correlated with rFA, FDi and rFDi (correlation coefficients (r) were 0.615, 0.643 and 0.567 for rFA, FDi and rFDi, respectively). The rFA values between grades (2&3, 2&4, 2&5, 3&5, 4&5) of muscle strength were significantly different (P<0.05), the rFDi values between grades (2&4, 2&5, 3&4, 3&5) of muscle strength were significantly different (P<0.05), while the rFA and rFDi values in the remaining groups of muscle strength grades showed no significant differences(P>0.05).

Conclusions: Preoperative DTI and diffusion tensor tractography may quantify the injury degrees of CST and the extent of motor dysfunction in patients with brain glioma.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182795PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568729PMC
October 2017

Sexually dimorphic traits in the silkworm, Bombyx mori, are regulated by doublesex.

Insect Biochem Mol Biol 2017 01 17;80:42-51. Epub 2016 Nov 17.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The DM domain genes, doublesex (dsx) in insects, or their structural homologs, male abnormal 3 (mab-3) in nematodes and Dmrt1 (doublesex and mab-3-related transcription factor 1) in mammals, are downstream regulators of the sex determination pathway that control sexually dimorphic development. Despite the functional importance of dsx and its potential applications in sterile insect technologies (SITs), the mechanisms by which it controls sexually dimorphic traits and the subsequent developmental gene networks in insects are poorly understood. Phylogenetic analyses indicate that insect dsx genes have sex-specific alternative splicing isoforms, whereas other taxa do not. We exploited genome editing and transgenesis technologies to induce mutations in either the male-specific isoform (dsx) or common region (dsx) of dsx in the somatic tissues of the lepidopteran model insect Bombyx mori. Disruptions of gene function produced either male-specific sexually-dimorphic defects or intersexual phenotypes; these results differ from those observed in other insects, including Drosophila melanogaster. Our data provide insights into the divergence of the insect sex determination pathways related to the most conserved downstream component dsx.
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http://dx.doi.org/10.1016/j.ibmb.2016.11.005DOI Listing
January 2017

Identification and Characterization of Erysiphe necator-Responsive MicroRNAs in Chinese Wild Vitis pseudoreticulata by High-Throughput Sequencing.

Front Plant Sci 2016 31;7:621. Epub 2016 May 31.

State Key Laboratory of Crop Stress Biology for Arid Areas, College of Horticulture, Northwest A&F UniversityYangling, China; College of Horticulture, Northwest A&F UniversityYangling, China; Key Laboratory of Horticultural Plant Biology and Germplasm Innovation in Northwest China, Ministry of Agriculture, College of Horticulture, Northwest A& F UniversityYangling, China.

Grapevine powdery mildew is one of the most damaging fungal diseases. Therefore, a precise understanding of the grapevine disease resistance system becomes a subject of significant importance. Plant microRNAs(miRNAs) have been implicated to play regulatory roles in plant biotic stress responses. In this study, high-throughput sequencing and miRDeep-P were employed to identify miRNAs in Chinese wild Vitis pseudoreticulata leaves following inoculation with Erysiphe necator. Altogether, 126 previously identified microRNAs and 124 novel candidates of miRNA genes were detected. Among them, 43 conserved miRNAs belong to 20 families and 23 non-conserved but previously-known miRNAs belong to 15 families. Following E. necator inoculation, 119 miRNAs were down-regulated and 131 were up-regulated. Furthermore, the expression changes occurring in 32 miRNAs were significant. The expression patterns of some miRNAs were validated by semi-quantitative RT-PCR and qRT-PCR. A total of 485 target genes were predicted and categorized by Gene Ontology (GO). In addition, 14 vvi-miRNAs were screened with 36 targets which may be involved in powdery mildew resistance in grape. Highly accumulated vvi-NewmiR2118 was detected from accession "Baihe-35-1," whose targets were mostly NBS-LRR resistance genes. It was down-regulated rapidly and strongly in "Baihe-35-1" leaves after inoculated with E. necator, indicating its involvement in grape powdery mildew resistance. Finally, the study verified interaction between vvi-NewmiR2118 and RPP13 by histochemical staining and GUS fluorescence quantitative assay.
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http://dx.doi.org/10.3389/fpls.2016.00621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4885880PMC
June 2016

Expansion of CRISPR targeting sites in Bombyx mori.

Insect Biochem Mol Biol 2016 May 24;72:31-40. Epub 2016 Mar 24.

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China. Electronic address:

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.
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http://dx.doi.org/10.1016/j.ibmb.2016.03.006DOI Listing
May 2016

Ectopic expression of ecdysone oxidase impairs tissue degeneration in Bombyx mori.

Proc Biol Sci 2015 Jun;282(1809):20150513

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, People's Republic of China

Metamorphosis in insects includes a series of programmed tissue histolysis and remolding processes that are controlled by two major classes of hormones, juvenile hormones and ecdysteroids. Precise pulses of ecdysteroids (the most active ecdysteroid is 20-hydroxyecdysone, 20E), are regulated by both biosynthesis and metabolism. In this study, we show that ecdysone oxidase (EO), a 20E inactivation enzyme, expresses predominantly in the midgut during the early pupal stage in the lepidopteran model insect, Bombyx mori. Depletion of BmEO using the transgenic CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/RNA-guided Cas9 nucleases) system extended the duration of the final instar larval stage. Ubiquitous transgenic overexpression of BmEO using the Gal4/UAS system induced lethality during the larval-pupal transition. When BmEO was specifically overexpressed in the middle silk gland (MSG), degeneration of MSG at the onset of metamorphosis was blocked. Transmission electron microscope and LysoTracker analyses showed that the autophagy pathway in MSG is inhibited by BmEO ectopic expression. Furthermore, RNA-seq analysis revealed that the genes involved in autophagic cell death and the mTOR signal pathway are affected by overexpression of BmEO. Taken together, BmEO functional studies reported here provide insights into ecdysone regulation of tissue degeneration during metamorphosis.
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http://dx.doi.org/10.1098/rspb.2015.0513DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4590451PMC
June 2015

MiR-2 family targets awd and fng to regulate wing morphogenesis in Bombyx mori.

RNA Biol 2015 ;12(7):742-8

a Faculty of Life Sciences; Northwestern Polytechnical University ; Xi'an , China.

MicroRNAs (miRNAs) are post-transcriptional regulators that target specific mRNAs for repression and thus play key roles in many biological processes, including insect wing morphogenesis. miR-2 is an invertebrate-specific miRNA family that has been predicted in the fruit fly, Drosophila melanogaster, to be involved in regulating the Notch signaling pathway. We show here that miR-2 plays a critical role in wing morphogenesis in the silkworm, Bombyx mori, a lepidopteran model insect. Transgenic over-expression of a miR-2 cluster using a Gal4/UAS system results in deformed adult wings, supporting the conclusion that miR-2 regulates functions essential for normal wing morphogenesis. Two genes, abnormal wing disc (awd) and fringe (fng), which are positive regulators in Notch signaling, are identified as miR-2 targets and validated by a dual-luciferase reporter assay. The relative abundance of both awd and fng expression products was reduced significantly in transgenic animals, implicating them in the abnormal wing phenotype. Furthermore, somatic mutagenesis analysis of awd and fng using the CRISPR/Cas9 system and knock-out mutants also resulted in deformed wings similar to those observed in the miR-2 overexpression transgenic animals. The critical role of miR-2 in Bombyx wing morphogenesis may provide a potential target in future lepidopteran pest control.
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http://dx.doi.org/10.1080/15476286.2015.1048957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4615647PMC
March 2016

Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.

Int J Biol Sci 2015 5;11(2):176-85. Epub 2015 Jan 5.

1. Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

RNA interference has been described as a powerful genetic tool for gene functional analysis and a promising approach for pest management. However, RNAi efficiency varies significantly among insect species due to distinct RNAi machineries. Lepidopteran insects include a large number of pests as well as model insects, such as the silkworm, Bombyx mori. However, only limited success of in vivo RNAi has been reported in lepidoptera, particularly during the larval stages when the worms feed the most and do the most harm to the host plant. Enhancing the efficiency of larval RNAi in lepidoptera is urgently needed to develop RNAi-based pest management strategies. In the present study, we investigate the function of the conserved RNAi core factor, Argonaute2 (Ago2), in mediating B. mori RNAi efficiency. We demonstrate that introducing BmAgo2 dsRNA inhibits the RNAi response in both BmN cells and embryos. Furthermore, we establish several transgenic silkworm lines to assess the roles of BmAgo2 in larval RNAi. Over-expressing BmAgo2 significantly facilitated both dsRNA-mediated larval RNAi when targeting DsRed using dsRNA injection and shRNA-mediated larval RNAi when targeting BmBlos2 using transgenic shRNA expression. Our results show that BmAgo2 is involved in RNAi in B. mori and provides a promising approach for improving larval RNAi efficiency in B. mori and in lepidopteran insects in general.
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http://dx.doi.org/10.7150/ijbs.10235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279093PMC
September 2015