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Wang H, Xie H, Zhong A, Xie Q. Efficient production of the high-intensity natural sweetener siamenoside I by the exo-1,3-beta glucanase (Exo15) from Meyerozyma guilliermondii LHGNSJ-VS01. 3 Biotech 2025; 15:94. [PMID: 40124134 PMCID: PMC11926301 DOI: 10.1007/s13205-025-04260-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2024] [Accepted: 03/02/2025] [Indexed: 03/25/2025] Open
Abstract
The scarcity of siamenoside I (SI) hindered its widespread application. Addressing this challenge, we devised an innovative biocatalytic strategy and biological solution for large-scale SI production. Endo 15, an endophyte from Siraitia grosvenorii, exhibited excellent proficiency in SI synthesis, achieving a remarkable 50.65% SI abundance. By harnessing the extracellular protein of Endo 15 (EP), we further escalated SI abundance to 83.59 ± 2.5%, accompanied by full substrate conversion. Delving into the underlying mechanisms, we identified Exo15, a distinct functional protein derived from EP, displaying merely 48.88% amino acid similarity to the yeast exo-1,3-beta glucanase (Exg1). Successfully overexpressing Exo15 in E. coli, we confirmed its functionality in line with EP. Exo15 exhibited exceptional catalytic prowess, efficiently hydrolyzing mogroside V into the high-potency sweetener SI, with unparalleled activity and specificity. Our groundbreaking approach yielded an impressive SI titer of 54 g/L, coupled with an average conversion rate of 2.5 g/L per hour. These outstanding outcomes underscore the immense potential of Exo15 in cost-effective industrial production of the premium natural sweetener, siamenoside I, paving the way for its widespread adoption. Supplementary Information The online version contains supplementary material available at 10.1007/s13205-025-04260-2.
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Affiliation(s)
- Hongjiang Wang
- Bioengineering Center, Chengdu Biopurify Phytochemicals Ltd, 2F, No.11 Building, No.388 Rongtaidadao CNSTP, Wenjiang Zone, Chengdu, 611130 Sichuan China
- Synthetic Biology Laboratory, Chengdu Greenpure Biopharma co., Ltd, 501 #, Building 1, No.269 Fenghuang Road, Shuangliu District, Chengdu, 610219 Sichuan China
| | - Haifeng Xie
- Bioengineering Center, Chengdu Biopurify Phytochemicals Ltd, 2F, No.11 Building, No.388 Rongtaidadao CNSTP, Wenjiang Zone, Chengdu, 611130 Sichuan China
| | - Ailing Zhong
- Bioengineering Center, Chengdu Biopurify Phytochemicals Ltd, 2F, No.11 Building, No.388 Rongtaidadao CNSTP, Wenjiang Zone, Chengdu, 611130 Sichuan China
- Synthetic Biology Laboratory, Chengdu Greenpure Biopharma co., Ltd, 501 #, Building 1, No.269 Fenghuang Road, Shuangliu District, Chengdu, 610219 Sichuan China
| | - Qilin Xie
- Bioengineering Center, Chengdu Biopurify Phytochemicals Ltd, 2F, No.11 Building, No.388 Rongtaidadao CNSTP, Wenjiang Zone, Chengdu, 611130 Sichuan China
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Qiao X, Sun X, Wang S, Zhai C, Tang W, Tang T, Zhang J, He Z. Characterization of Flexusin A, a Novel Circular Bacteriocin Produced by Marine Bacterium Bacillus flexus R29-2. Mar Drugs 2025; 23:95. [PMID: 40137281 PMCID: PMC11943950 DOI: 10.3390/md23030095] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2025] [Revised: 02/14/2025] [Accepted: 02/18/2025] [Indexed: 03/27/2025] Open
Abstract
Circular bacteriocins are potent antimicrobials against pathogenic Gram-positives. In searching for marine bacteriocins, an antibacterial peptide (flexusin A) was purified from the fermentation broth of marine bacterium Bacillus flexus R29-2. Genome sequencing and gene annotation revealed the chromosome contained an unknown circular bacteriocin gene cluster. Approaches including shot-gun proteomics analysis, AntiSMASH and BAGEL4 predication as well as the comprehensive sequence alignment, were then conducted, respectively, to verify the correlation of flexusin A with the gene-encoded precursor peptide. The results confirmed that flexusin A was the mature circular bacteriocin of the predicated precursor peptide with six amino acids as leader peptide. Flexusin A was 6098.4 Da in size, with a net charge of +3 and PI of 9.60. It shared the typical saposin-like fold spatial conformation features as commonly found in other circular bacteriocins. Flexusin A was pH, thermal, and protease tolerant. It exhibited a narrow antimicrobial spectrum against Gram-positives, and it can strongly inhibit Staphylococcus aureus by causing cell destruction via membrane destabilization. Taken together, a novel circular bacteriocin flexusin A was identified in this work. The characterization of flexusin A has extended circular bacteriocins family to 26 members. This is also the first report on bacteriocin production by B. flexus.
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Affiliation(s)
- Xiaoni Qiao
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
| | - Xiaowen Sun
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
| | - Shuting Wang
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
| | - Chen Zhai
- State Key Laboratory of Animal Nutrition, Institute of Animal Sciences of Chinese Academy of Agricultural Sciences, Beijing 100193, China;
| | - Wei Tang
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
- Marine Microbial Engineering Research and Development Center, Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China
| | - Tao Tang
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
- Marine Microbial Engineering Research and Development Center, Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China
| | - Jun Zhang
- School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China; (X.Q.); (X.S.); (S.W.); (W.T.); (T.T.); (J.Z.)
| | - Zengguo He
- Qingdao Bioantai Biotechnology Co., Ltd., Qingdao 266000, China
- Marine Microbial Engineering Research and Development Center, Marine Biomedical Research Institute of Qingdao, Qingdao 266071, China
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3
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Xiao Y, Zhao Z, Zhang T, Xu X, Anik K, Qiu Y, Xu Z, Li S, Xu H. A new glycoprotein from pigeon egg: Study on its structure and digestive characteristics. Food Res Int 2024; 194:114875. [PMID: 39232513 DOI: 10.1016/j.foodres.2024.114875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 07/31/2024] [Accepted: 08/05/2024] [Indexed: 09/06/2024]
Abstract
Pigeon egg white (PEW) is widely recognized as a promising source of bioactive proteins, with a high degree of glycosylation. This study focused on the characterization of a novel glycoprotein extracted from PEW, known as ovalbumin-related protein Y (OVAY). Our investigation included an analysis of the N-glycan and protein structures of OVAY, as well as an examination of simulated gastrointestinal digestive products and the transmembrane transport mechanism of OVAY-digested peptides. The results revealed that OVAY contains two glycosylation sites (Asn 62, 215) and consists of 30 N-linked glycoforms, with the top three glycans being N6H3, N6H7S1, and N6H5. Additionally, OVAY is rich in Gal and sialic acid and exhibits a rod-like molecular structure. Furthermore, it was found that OVAY demonstrates resistance to gastric digestion, with its digested peptides primarily transported via PepT1 and endocytosis. This study provides insight into the glycoprotein structure of OVAY and elucidates its physiological activity, providing a theoretical reference for the development of a novel sialate-rich protein.
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Affiliation(s)
- Yu Xiao
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Zeyun Zhao
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Tao Zhang
- College of Food Science and Engineering, Nanjing University of Finance and Economics, Nanjing 210023, PR China
| | - Xiaoqi Xu
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China.
| | - Khan Anik
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Yibin Qiu
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Zheng Xu
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Sha Li
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
| | - Hong Xu
- College of Food and Light Industry, Nanjing Tech University, Nanjing 211800, PR China
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Zhang ZT, Wang H, Dong H, Cong B. Comparative hemolymph proteomic analyses of the freezing and resistance-freezing Ostrinia furnacalis (Guenée). Sci Rep 2024; 14:2580. [PMID: 38297109 PMCID: PMC10830562 DOI: 10.1038/s41598-024-52792-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Accepted: 01/23/2024] [Indexed: 02/02/2024] Open
Abstract
The Asian corn borer, Ostrinia furnacalis (Guenée) (Lepidoptera: Crambidae), is one of the most harmful pests of maize in Asia. It poses a significant threat to maize production, causing economic losses due to its strong ecological adaptation. In this study, we compared and analyzed the hemolymph proteome between freezing and resistance-freezing O. furnacalis strains using two-dimensional gel electrophoresis to gain insights into the mechanisms of cold resistance. The results revealed that 300-400 hemolymph protein spots were common, with 24 spots showing differences between the two strains. Spectrometry analysis revealed 21 protein spots, including 17 upregulated spots and 4 downregulated ones. The expression of upregulation/downregulation proteins plays a crucial role in the metabolism, energy supply, and defense reaction of insects. Proteomics research not only provides a method for investigating protein expression patterns but also identifies numerous attractive candidates for further exploration.
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Affiliation(s)
- Zhu-Ting Zhang
- Shenyang Agricultural University, Shenyang, 110866, Liaoning, People's Republic of China
- Kaili University, 556011, Kaili, People's Republic of China
| | - Huan Wang
- Shenyang Agricultural University, Shenyang, 110866, Liaoning, People's Republic of China.
| | - Hui Dong
- Shenyang Agricultural University, Shenyang, 110866, Liaoning, People's Republic of China.
| | - Bin Cong
- Shenyang Agricultural University, Shenyang, 110866, Liaoning, People's Republic of China
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Nishida H, Kanao E, Ishihama Y. Centrifugal Gel Crushing Tips for Gel-Based Proteome Analysis. Anal Chem 2023; 95:18311-18315. [PMID: 38055789 DOI: 10.1021/acs.analchem.3c02527] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/08/2023]
Abstract
We have developed a centrifugal gel-crushing method using a pipet tip. Polyacrylamide gel slices are extruded from the narrowing cavity of a pipet tip by centrifugation in a few minutes to crush them into pieces of appropriate size. The size of the crushed gel could be controlled by several parameters, including centrifugal force and pipet tip cavity. In shotgun proteomics, gel-based LC/MS/MS, so-called GeLC/MS/MS, involves the essential but tedious processes of prefractionation by SDS-PAGE, followed by dicing the entire gel lane into several parts, fine dicing, and in-gel digestion after the diced gel is manually transferred to a microtube. In this study, we developed an alternative way to crush the prefractionated gel slice into optionally small and irregular-shaped gels by centrifugal extrusion of the sliced gel from the narrow cavity of a pipet tip. As a result, we observed improved recovery and reproducibility of digested proteins compared to the conventional method of manual dicing. We believe that this simple and rapid method of crushing polyacrylamide gels, which allows for parallel operations and automation, is useful for GeLC/MS/MS analysis and applicable to other approaches, including top-down proteomics.
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Affiliation(s)
- Hiroshi Nishida
- Division of Medicinal Frontier Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
| | - Eisuke Kanao
- Division of Medicinal Frontier Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
- Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan
| | - Yasushi Ishihama
- Division of Medicinal Frontier Sciences, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
- Laboratory of Clinical and Analytical Chemistry, National Institute of Biomedical Innovation, Health and Nutrition, Ibaraki, Osaka 567-0085, Japan
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Hao T, Xu D, Cao X, Chen Q, Chen F, Liu Q, Tang Y, Zhou Y, Li Y, Mai K, Ai Q. Regulation of low-density lipoprotein on lipid metabolism in macrophages of large yellow croaker (Larimichthys crocea). Biochim Biophys Acta Mol Cell Biol Lipids 2023; 1868:159397. [PMID: 37741313 DOI: 10.1016/j.bbalip.2023.159397] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2023] [Revised: 09/15/2023] [Accepted: 09/20/2023] [Indexed: 09/25/2023]
Abstract
Low-density lipoprotein (LDL) is the main carrier of cholesterol transport in plasma, which participates in regulating lipid homeostasis. Studies in mammals have shown that high levels of LDL in plasma absorbed by macrophages trigger the formation of lipid-rich foam cells, leading to the development of atherosclerotic plaques. Although lipid-rich atherosclerosis-like lesions have been discovered in the aorta of several fish species, the physiological function of LDL in fish macrophages remains poorly understood. In the present study, LDL was isolated from the plasma of large yellow croaker (Larimichthys crocea), and mass spectrometry analysis identified two truncated forms of apolipoprotein B100 in the LDL protein profile. Transcriptomic analysis of LDL-stimulated macrophages revealed that differentially expressed genes (DEGs) were enriched in various pathways related to lipid metabolism, as confirmed by the fact that LDL increased total cholesterol and cholesteryl esters content. Meanwhile, the gene and protein expression levels of perilipin2 (PLIN2), a DEG enriched in the PPAR signaling pathway, were upregulated in response to LDL stimulation. Importantly, knocking down plin2 significantly attenuates LDL-induced cholesterol accumulation and promotes cholesterol efflux. Furthermore, the transcription factor PPARγ, which is upregulated in response to LDL stimulation, can enhance the promoter activity of plin2. In conclusion, this study suggests that LDL may upregulate plin2 expression through PPARγ, resulting in cholesterol accumulation in fish macrophages. This study will facilitate the investigation of the function of LDL in regulating lipid homeostasis in macrophages and shed light on the evolutionary origin of LDL metabolism in vertebrates.
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Affiliation(s)
- Tingting Hao
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Dan Xu
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Xiufei Cao
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Qiuchi Chen
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Fan Chen
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Qiangde Liu
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Yuhang Tang
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Yan Zhou
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Yueru Li
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China
| | - Kangsen Mai
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, 266237 Qingdao, Shandong, People's Republic of China
| | - Qinghui Ai
- Key Laboratory of Aquaculture Nutrition and Feed (Ministry of Agriculture and Rural Affairs) and Key Laboratory of Mariculture (Ministry of Education), Ocean University of China, 5 Yushan Road, 266003 Qingdao, Shandong, People's Republic of China; Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, 1 Wenhai Road, 266237 Qingdao, Shandong, People's Republic of China.
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7
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Li X, Chen L, Wang Y, Guo X, He ZG. Zinc excess impairs Mycobacterium bovis growth through triggering a Zur-IdeR-iron homeostasis signal pathway. Microbiol Spectr 2023; 11:e0106923. [PMID: 37668384 PMCID: PMC10580935 DOI: 10.1128/spectrum.01069-23] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Accepted: 07/15/2023] [Indexed: 09/06/2023] Open
Abstract
Zinc excess is toxic to bacteria and, thus, represents an important innate defense mechanism of host cells, especially against mycobacterial infections. However, the signaling pathway triggered by zinc excess and its relationship with iron homeostasis remain poorly understood in mycobacteria. Here, we characterize a novel Zur-IdeR-iron homeostasis signaling pathway that modulates the growth of Mycobacterium bovis under zinc toxicity. We found that the regulator Zur interacts with the iron-homeostasis regulator IdeR, enhancing the DNA-binding ability of IdeR. Excess zinc disrupts this interaction and represses ideR transcription through Zur, which promotes the expression of iron uptake genes and leads to the accumulation of intracellular iron in M. bovis. The elevated iron levels lower the bacterial survival ability under excess zinc stress. Consistently, deleting zur hinders intracellular iron accumulation of M. bovis and enhances bacterial growth under stress, while silencing ideR impairs the growth of the wild-type and zur-deleted strains under the same conditions. Interestingly, both Zur and IdeR are conserved in bacteria facing zinc toxicity. Overall, our work uncovers a novel antimicrobial signal pathway whereby zinc excess disrupts iron homeostasis, which may deepen our understanding of the crosstalk mechanism between iron and zinc homeostasis in bacteria.IMPORTANCEAs a catalytic and structural cofactor of proteins, zinc is essential for almost all living organisms. However, zinc excess is toxic and represents a vital innate immunity strategy of macrophages to combat intracellular pathogens, especially against mycobacterial pathogens such as Mycobacterium tuberculosis, the causative agent of tuberculosis. Here, we first characterize an antibacterial signaling pathway of zinc excess and its relationship with iron homeostasis in M. bovis. We found that excess zinc inhibits the transcription of ideR and its DNA-binding activity through Zur, which, in turn, promotes the expression of iron uptake genes, causes intracellular iron accumulation, and finally impairs the bacterial growth. This study reveals the existence of the Zur-IdeR-iron homeostasis pathway triggered by zinc excess in M. bovis, which will shed light on the crosstalk mechanisms between zinc and iron homeostasis in bacteria and the antimicrobial mechanisms of host-mediated zinc toxicity.
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Affiliation(s)
- Xiaohui Li
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China
| | - Liu Chen
- College of Life Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Yuankun Wang
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China
| | - Xiao Guo
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China
| | - Zheng-Guo He
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi Research Center for Microbial and Enzyme Engineering Technology, College of Life Science and Technology, Guangxi University, Nanning, Guangxi, China
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Maurer J, Grouzmann E, Eugster PJ. Tutorial review for peptide assays: An ounce of pre-analytics is worth a pound of cure. J Chromatogr B Analyt Technol Biomed Life Sci 2023; 1229:123904. [PMID: 37832388 DOI: 10.1016/j.jchromb.2023.123904] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2023] [Revised: 10/04/2023] [Accepted: 10/05/2023] [Indexed: 10/15/2023]
Abstract
The recent increase in peptidomimetic-based medications and the growing interest in peptide hormones has brought new attention to the quantification of peptides for diagnostic purposes. Indeed, the circulating concentrations of peptide hormones in the blood provide a snapshot of the state of the body and could eventually lead to detecting a particular health condition. Although extremely useful, the quantification of such molecules, preferably by liquid chromatography coupled to mass spectrometry, might be quite tricky. First, peptides are subjected to hydrolysis, oxidation, and other post-translational modifications, and, most importantly, they are substrates of specific and nonspecific proteases in biological matrixes. All these events might continue after sampling, changing the peptide hormone concentrations. Second, because they include positively and negatively charged groups and hydrophilic and hydrophobic residues, they interact with their environment; these interactions might lead to a local change in the measured concentrations. A phenomenon such as nonspecific adsorption to lab glassware or materials has often a tremendous effect on the concentration and needs to be controlled with particular care. Finally, the circulating levels of peptides might be low (pico- or femtomolar range), increasing the impact of the aforementioned effects and inducing the need for highly sensitive instruments and well-optimized methods. Thus, despite the extreme diversity of these peptides and their matrixes, there is a common challenge for all the assays: the need to keep concentrations unchanged from sampling to analysis. While significant efforts are often placed on optimizing the analysis, few studies consider in depth the impact of pre-analytical steps on the results. By working through practical examples, this solution-oriented tutorial review addresses typical pre-analytical challenges encountered during the development of a peptide assay from the standpoint of a clinical laboratory. We provide tips and tricks to avoid pitfalls as well as strategies to guide all new developments. Our ultimate goal is to increase pre-analytical awareness to ensure that newly developed peptide assays produce robust and accurate results.
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Affiliation(s)
- Jonathan Maurer
- Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Eric Grouzmann
- Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Philippe J Eugster
- Service of Clinical Pharmacology, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
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9
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Dewi DNSS, Mertaniasih NM, Soedarsono, Hagino K, Yamazaki T, Ozeki Y, Artama WT, Kobayashi H, Inouchi E, Yoshida Y, Ishikawa S, Shaban AK, Tateishi Y, Nishiyama A, Ato M, Matsumoto S. Antibodies against native proteins of Mycobacterium tuberculosis can detect pulmonary tuberculosis patients. Sci Rep 2023; 13:12685. [PMID: 37542102 PMCID: PMC10403504 DOI: 10.1038/s41598-023-39436-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Accepted: 07/25/2023] [Indexed: 08/06/2023] Open
Abstract
Accurate point-of-care testing (POCT) is critical for managing tuberculosis (TB). However, current antibody-based diagnosis shows low specificity and sensitivity. To find proper antigen candidates for TB diagnosis by antibodies, we assessed IgGs responsiveness to Mycobacterium tuberculosis proteins in pulmonary TB (PTB) patients. We employed major secreted proteins, such as Rv1860, Ag85C, PstS1, Rv2878c, Ag85B, and Rv1926c that were directly purified from M. tuberculosis. In the first screening, we found that IgG levels were significantly elevated in PTB patients only against Rv1860, PstS1, and Ag85B among tested antigens. However, recombinant PstS1 and Ag85B from Escherichia coli (E. coli) couldn't distinguish PTB patients and healthy controls (HC). Recombinant Rv1860 was not checked due to its little expression. Then, the 59 confirmed PTB patients from Soetomo General Academic Hospital, Surabaya, Indonesia, and 102 HC were tested to Rv1860 and Ag85B only due to the low yield of the PstS1 from M. tuberculosis. The ROC analysis using native Ag85B and Rv1860 showed an acceptable area under curve for diagnosis, which is 0.812 (95% CI 0.734-0.890, p < 0.0001) and 0.821 (95% CI 0.752-0.890, p < 0.0001). This study indicates that taking consideration of native protein structure is key in developing TB's POCT by antibody-based diagnosis.
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Affiliation(s)
- Desak Nyoman Surya Suameitria Dewi
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan.
- Department of Microbiology, Faculty of Medicine, Universitas Ciputra, CitraLand CBD Boulevard, Made, Kec. Sambikerep, Surabaya, 60219, Indonesia.
| | - Ni Made Mertaniasih
- Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Jl. Mayjen Prof. Dr. Moestopo 47, Surabaya, 60131, Indonesia.
- Laboratory of Tuberculosis, Institute of Tropical Disease, Universitas Airlangga, Kampus C Jl. Mulyorejo, Surabaya, 60115, Indonesia.
| | - Soedarsono
- Sub-Pulmonology Department of Internal Medicine, Faculty of Medicine, Hang Tuah University, Komplek Barat RSAL Dr. Ramelan, Jl. Gadung No.1, Jagir, Surabaya, 60111, Indonesia
| | - Kimika Hagino
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Tomoya Yamazaki
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Yuriko Ozeki
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Wayan Tunas Artama
- Department of Biochemistry, Faculty of Veterinary Medicine, Universitas Gadjah Mada, Jl. Fauna 2 Karangmalang, Yogyakarta, 55281, Indonesia
- One Health/Eco-Health Resource Center, Universitas Gadjah Mada, Jl. Teknika Utara, Barek, Sleman, Yogyakarta, 55281, Indonesia
| | - Haruka Kobayashi
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Erina Inouchi
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Yutaka Yoshida
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Satoshi Ishikawa
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
- Fukuyama Zoo, 276‑1, Fukuda, Ashida‑cho, Fukuyama, Hiroshima, 720‑1264, Japan
| | - Amina Kaboso Shaban
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Yoshitaka Tateishi
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Akihito Nishiyama
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan
| | - Manabu Ato
- Department of Mycobacteriology, Leprosy Research Center, National Institute of Infectious Diseases, Aoba-cho 4-2-1, Higashimurayama-shi, Tokyo, 189-0002, Japan
| | - Sohkichi Matsumoto
- Department of Bacteriology, School of Medicine, Niigata University, Asahimachi-Dori 1-757, Chuo-ku, Niigata, 951-8510, Japan.
- Department of Medical Microbiology, Faculty of Medicine, Universitas Airlangga, Jl. Mayjen Prof. Dr. Moestopo 47, Surabaya, 60131, Indonesia.
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10
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Shao L, Chen S, Ning Z, Xu X, Wang H. Characterization of effector protein Hap determining meat spoilage process from meat-borne Aeromonas salmonicida. Food Chem 2023; 410:135457. [PMID: 36641914 DOI: 10.1016/j.foodchem.2023.135457] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2022] [Revised: 11/21/2022] [Accepted: 01/09/2023] [Indexed: 01/13/2023]
Abstract
The spoilage roles of effector proteins secreted by dominant spoilage bacteria during food spoilage remained unknown. In this investigation, a hemagglutinin protease (Hap) belonging to the M4 family metallopeptidase was identified from Aeromonas salmonicida 29 isolate. It, has a molecular weight of 33.5 kDa, a Vmax of 17.06 μg/mL/min, and a Km of 2.46 mg/mL, and is conserved in various dominant spoilage bacteria. The stability testing demonstrated that Hap could maintain specific activity in the common environments (pH, temperature, and metal ions) of chilled meat. It exhibited high spoilage ability on meat in situ, increasing TVB-N, pH values, and the production of volatile organic compounds (VOCs), which was consistent with proteolytic activity analysis, completely confirming the determinant role of Hap for meat spoilage. These observations will enrich the spoilage theory and provide new insights into the control of food quality and safety.
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Affiliation(s)
- Liangting Shao
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Shanshan Chen
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Zhenzhen Ning
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Xinglian Xu
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China
| | - Huhu Wang
- Jiangsu Collaborative Innovation Center of Meat Production and Processing, Quality and Safety Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, PR China.
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11
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Li Y, Zhang Z, Wang J, Shan Y, Tian H, Cui P, Ru S. Zebrafish (Danio rerio) TRβ- and TTR-based electrochemical biosensors: Construction and application for the evaluation of thyroid-disrupting activity of bisphenols. ENVIRONMENTAL POLLUTION (BARKING, ESSEX : 1987) 2023; 330:121745. [PMID: 37127237 DOI: 10.1016/j.envpol.2023.121745] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/01/2023] [Revised: 04/19/2023] [Accepted: 04/28/2023] [Indexed: 05/03/2023]
Abstract
Thyroid-disrupting chemicals (TDCs) have received increasing concerns because of their negative health impacts on both wildlife and humans. This study aimed to develop in vitro screening assays for TDCs based on thyroid hormone receptor β (TRβ) and transthyretin (TTR) proteins. Firstly, the recombinant ligand-binding domain of TRβ (TRβ-LBD) and TTR proteins of zebrafish were produced by eukaryotic expression system and then used as bio-recognition components to construct electrochemical biosensors. In the biosensors, the supported bilayer lipid membrane (s-BLM) was used as a matrix to immobilize proteins, and gold nanoflowers (AuNFs) were used to improve the sensitivity by increasing electroactive surface area. Under the optimizing conditions, the zfTRβ-LBD/AuNFs/s-BLM/GCE biosensor had a detection range of 0.23 nM-1.92 μM and a detection limit of 0.07 nM for triiodothyronine (T3), while the zfTTR/AuNFs/s-BLM/GCE biosensor had a detection range of 0.46 nM-3.84 μM, with a detection limit of 0.13 nM. Based on the constructed biosensors, the order of T3 equivalent concentrations of bisphenols was BPA ≈ BPS > BPF > BPAF ≈ BPAP > BPZ, which was similar to the results of recombinant TRβ two-hybrid yeast assay. Furthermore, the reliability of the biosensors was validated by molecular docking, in which BPA and BPS showed higher binding affinity to zfTRβ-LBD. Therefore, this study provided a valuable tool for efficiently screening TDCs.
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Affiliation(s)
- Yuejiao Li
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China
| | - Zhenzhong Zhang
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China
| | - Jun Wang
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China
| | - Yeqi Shan
- CAS and Shandong Province Key Laboratory of Experimental Marine Biology & Center of Deep Sea Research, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China
| | - Hua Tian
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China
| | - Pengfei Cui
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China
| | - Shaoguo Ru
- College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao, 266003, Shandong, China.
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12
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Establishment and application of multiple immunoassays for environmental estrogens based on recombinant Japanese flounder (Paralichthys olivaceus) choriogenin protein. Talanta 2023; 254:124135. [PMID: 36470019 DOI: 10.1016/j.talanta.2022.124135] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2022] [Revised: 11/21/2022] [Accepted: 11/23/2022] [Indexed: 11/27/2022]
Abstract
Environmental estrogens have generated great concern because of their potential threat to aquatic organisms; however, the commonly used vitellogenin (Vtg) biomarker detection methods are not capable of detecting estrogenic activity below 10 ng/L 17β-estradiol. In this study, we developed multiple immunoassays based on Japanese flounder (Paralichthys olivaceus) choriogenin (Chg), a highly sensitive biomarker of environmental estrogens. Chg genes (ChgL and ChgH) of Japanese flounder were cloned for the first time, and a recombinant ChgL protein with a molecular weight of approximately 52 kDa was prepared using a prokaryotic expression system and purified using Ni-affinity column chromatography. Subsequently, specific monoclonal antibodies against ChgL were prepared and used to develop sandwich enzyme-linked immunosorbent assays (ELISAs), which had a detection range of 3.9-250 ng/mL and detection limit of 1.9 ng/mL. An immunofluorescence method was also established and used to visually detect ChgL induction in the tissues. In addition, a lateral flow immunoassay for ChgL that could detect estrogen activity within 10 min was developed. Finally, the reliability of the immunoassays was examined by measuring ChgL induction in the plasma and tissues of Japanese flounder exposed to 0, 2, 10, and 50 ng/L 17α-ethinylestradiol (EE2). The results showed that 2 ng/L EE2 notably increased ChgL levels in the plasma, demonstrating that ChgL is more sensitive than Vtg to environmental estrogens; 50 ng/L EE2 induced obvious Chg induction in the sinusoidal vessels of the liver. Conclusions taken together, this study provides reliable methods for sensitive and rapid detection of estrogenic activity in aquatic environments.
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13
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Liepold T, Klafki HW, Kumar S, Walter J, Wirths O, Wiltfang J, Jahn O. Matrix Development for the Detection of Phosphorylated Amyloid-β Peptides by MALDI-TOF-MS. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2023; 34:505-512. [PMID: 36706152 PMCID: PMC9983008 DOI: 10.1021/jasms.2c00270] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Revised: 12/20/2022] [Accepted: 01/18/2023] [Indexed: 06/18/2023]
Abstract
Amyloid-β (Aβ) peptides, including post-translationally modified variants thereof, are believed to play a key role in the onset and progression of Alzheimer's disease. Suggested modified Aβ species with potential disease relevance include Aβ peptides phosphorylated at serine in position eight (pSer8-Aβ) or 26 (pSer26-Aβ). However, the published studies on those Aβ peptides essentially relied on antibody-based approaches. Thus, complementary analyses by mass spectrometry, as shown for other modified Aβ variants, will be necessary not only to unambiguously verify the existence of phosphorylated Aβ species in brain samples but also to reveal their exact identity as to phosphorylation sites and potential terminal truncations. With the aim of providing a novel tool for addressing this still-unresolved issue, we developed a customized matrix formulation, referred to as TOPAC, that allows for improved detection of synthetic phosphorylated Aβ species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. When TOPAC was compared with standard matrices, we observed higher signal intensities but minimal methionine oxidation and phosphate loss for intact pSer8-Aβ(1-40) and pSer26-Aβ(1-40). Similarly, TOPAC also improved the mass spectrometric detection and sequencing of the proteolytic cleavage products pSer8-Aβ(1-16) and pSer26-Aβ(17-28). We expect that TOPAC will facilitate future efforts to detect and characterize endogenous phosphorylated Aβ species in biological samples and that it may also find its use in phospho-proteomic approaches apart from applications in the Aβ field.
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Affiliation(s)
- Thomas Liepold
- Neuroproteomics
Group, Department of Molecular Neurobiology, Max Planck Institute for Multidisciplinary Sciences, 37075 Goettingen, Germany
| | - Hans-Wolfgang Klafki
- Department
of Psychiatry and Psychotherapy, University Medical Center Goettingen, Georg-August-University, 37075 Goettingen, Germany
| | - Sathish Kumar
- Department
of Neurology, University of Bonn, 53127 Bonn, Germany
| | - Jochen Walter
- Department
of Neurology, University of Bonn, 53127 Bonn, Germany
| | - Oliver Wirths
- Department
of Psychiatry and Psychotherapy, University Medical Center Goettingen, Georg-August-University, 37075 Goettingen, Germany
| | - Jens Wiltfang
- Department
of Psychiatry and Psychotherapy, University Medical Center Goettingen, Georg-August-University, 37075 Goettingen, Germany
| | - Olaf Jahn
- Neuroproteomics
Group, Department of Molecular Neurobiology, Max Planck Institute for Multidisciplinary Sciences, 37075 Goettingen, Germany
- Department
of Psychiatry and Psychotherapy, University Medical Center Goettingen, Georg-August-University, 37075 Goettingen, Germany
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14
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Miyamoto M, Himeda T, Ishihara K, Okuwa T, Kobayashi D, Nameta M, Karasawa Y, Chunhaphinyokul B, Yoshida Y, Tanaka N, Higuchi M, Komuro A. Theilovirus 3C Protease Cleaves the C-Terminal Domain of the Innate Immune RNA Sensor, Melanoma Differentiation-Associated Gene 5, and Impairs Double-Stranded RNA-Mediated IFN Response. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2023; 210:335-347. [PMID: 36525065 DOI: 10.4049/jimmunol.2200565] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 11/09/2022] [Indexed: 01/04/2023]
Abstract
Melanoma differentiation-associated gene 5 (MDA5), a member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), has pivotal roles in innate immune responses against many positive-stranded RNA viruses, including picornavirus and coronavirus. Upon engagement with dsRNA derived from viral infection, MDA5 initiates coordinated signal transduction leading to type I IFN induction to restrict viral replication. In this study, we describe a targeted cleavage events of MDA5 by the 3C protease from Theilovirus. Upon ectopic expression of theilovirus 3C protease from Saffold virus or Theiler's murine encephalomyelitis virus but not encephalomyocarditis virus, fragments of cleaved MDA5 were observed in a dose-dependent manner. When enzymatically inactive Theilovirus 3C protease was expressed, MDA5 cleavage was completely abrogated. Mass spectrometric analysis identified two cleavage sites at the C terminus of MDA5, cleaving off one of the RNA-binding domains. The same cleavage pattern was observed during Theilovirus infection. The cleavage of MDA5 by Theilovirus protease impaired ATP hydrolysis, RNA binding, and filament assembly on RNA, resulting in dysfunction of MDA5 as an innate immune RNA sensor for IFN induction. Furthermore, the cleavage-resistant MDA5 mutant against the 3C protease showed an enhanced IFN response during Saffold virus infection, indicating that Theilovirus has a strategy to circumvent the antiviral immune response by cleaving MDA5 using 3C protease. In summary, these data suggest MDA5 cleavage by 3C protease as a novel immune evasive strategy of Theilovirus.
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Affiliation(s)
- Masahiko Miyamoto
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
| | - Toshiki Himeda
- Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa, Japan
| | - Kazuki Ishihara
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
| | - Takako Okuwa
- Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa, Japan
| | - Daiki Kobayashi
- Omics Unit, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan
| | - Masaaki Nameta
- Electron Microscope Core Facility, Niigata University, Niigata, Japan
| | - Yu Karasawa
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
| | - Benyapa Chunhaphinyokul
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
| | - Yutaka Yoshida
- Department of Structural Pathology, Kidney Research Center, Niigata University, Niigata, Japan; and
| | - Nobuyuki Tanaka
- Division of Tumor Immunology, Miyagi Cancer Center Research Institute, Medeshima-Shiode, Natori, Miyagi, Japan
| | - Masaya Higuchi
- Department of Microbiology, Kanazawa Medical University School of Medicine, Ishikawa, Japan
| | - Akihiko Komuro
- Department of Biochemistry, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata, Japan
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15
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Abstract
In-gel digestion of protein spots derived from two-dimensional gels and their subsequent identification by mass spectrometry is involved in a multitude of mass spectrometry-driven proteomic experiments, including fluorescence two-dimensional difference gel electrophoresis (2D-DIGE). This type of proteomic methodology has been involved in the establishment of comparative proteome maps and in the identification of differentially expressed proteins and their isoforms in health and disease. Most in-gel digestion protocols follow a number of common steps including excision of the protein spots of interest, destaining, reduction and alkylation (for silver-stained gels), and dehydration and overnight digestion with the proteolytic enzyme of choice. While trypsin has been a mainstay of peptide digestion for many years, it does have its shortcomings, particularly related to incomplete peptide digestion, and this has led to a rise in popularity for other proteolytic enzymes either used alone or in combination. This chapter discusses the alternative enzymes available and describes the process of in-gel digestion using the enzyme trypsin.
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Affiliation(s)
| | - Kay Ohlendieck
- Department of Biology, Maynooth University, National University of Ireland, Maynooth, Co. Kildare, Ireland.
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16
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Guan Y, He J, Chen J, Li Y, Zhang X, Zheng Y, Jia L. Valorization of Fish Processing By-Products: Microstructural, Rheological, Functional, and Properties of Silver Carp Skin Type I Collagen. Foods 2022; 11:2985. [PMID: 36230061 PMCID: PMC9562877 DOI: 10.3390/foods11192985] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2022] [Revised: 09/20/2022] [Accepted: 09/21/2022] [Indexed: 11/17/2022] Open
Abstract
The objective of this study was to develop aquatic collagen production from fish processing by-product skin as a possible alternative to terrestrial sources. Silver carp skin collagen (SCSC) was isolated and identified as type I collagen, and LC-MS/MS analysis confirmed the SCSC as Hypophthalmichthys molitrix type I collagen, where the yield of SCSC was 40.35 ± 0.63% (dry basis weight). The thermal denaturation temperature (Td) value of SCSC was 30.37 °C, which was superior to the collagen of deep-sea fish and freshwater fish. Notably, SCSC had higher thermal stability than human placental collagen, and the rheological experiments showed that the SCSC was a shear-thinning pseudoplastic fluid. Moreover, SCSC was functionally superior to some other collagens from terrestrial sources, such as sheep, chicken cartilage, and pig skin collagen. Additionally, SCSC could provide a suitable environment for MC3T3-E1 cell growth and maintain normal cellular morphology. These results indicated that SCSC could be used for further applications in food, cosmetics, and biomedical fields.
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Affiliation(s)
- Yongxin Guan
- College of Chemistry and Chemical Engineering, Mudanjiang Normal University, Mudanjiang 157011, China
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Jianlin He
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Junde Chen
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Yushuang Li
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Xingkun Zhang
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Yan Zheng
- Technical Innovation Center for Utilization of Marine Biological Resources, Third Institute of Oceanography, Ministry of Natural Resources, Xiamen 361005, China
| | - Linyan Jia
- College of Chemistry and Chemical Engineering, Mudanjiang Normal University, Mudanjiang 157011, China
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17
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Purification, Identification and Neuroprotective Effects of Proteins from Bombyx batryticatus in Glu-Stimulated PC12 Cells. SEPARATIONS 2022. [DOI: 10.3390/separations9090236] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022] Open
Abstract
Bombyx batryticatus (BB) is one of the most commonly used Traditional Chinese Medicines (TCMs) in the treatment of convulsions and epilepsy. The antiepileptic effects of total proteins from BB (BBPs) have been proven in our previous research. In this study, BBPs were further purified, the neuroprotective effects were evaluated in Glu-stimulated PC12 cells, and the structure was identified by Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Six subfractions (PF-1 to PF-6) were obtained by DEAE-52 Sepharose FF ion-exchange chromatography. It was found that PF-1, PF-2, and PF-3, with similar protein compositions, possessed neuroprotective effects in Glu-stimulated PC12 cells by significantly increasing the GABA level, and decreasing the levels of IL-1β and TNF-α. The most active fraction (PF-2) was further separated by Sephadex G-75 gel filtration chromatography, and an effective protein component named PF-2-2 was obtained. Fluorescein isothiocyanate-labeled PF-2-2 (FITC-PF-2-2) was prepared, and the binding of FITC-F-2-2 to the PC12 cells was directly observed with a confocal microscope. PF-2-2 was found to first bind to the surface of PC12 cells and then internalize into the cells. The main band of PF-2-2 was then analyzed by MALDI-TOF/TOF-MS and searched in the MASCOT database; finally a protein named Low molecular mass 30 kDa lipoprotein 21G1 was identified. In conclusion, PF-2-2 and purified proteins isolated from BBPs have potential application prospects in the treatment of epilepsy.
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18
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Duan Y, Huang W, Zhan B, Li Y, Xu X, Li K, Li X, Liu X, Ding S, Wang S, Guo J, Wang Y, Gu Q. A Bioink Derived From Human Placenta Supporting Angiogenesis. Biomed Mater 2022; 17. [PMID: 35732166 DOI: 10.1088/1748-605x/ac7b5b] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2022] [Accepted: 06/22/2022] [Indexed: 11/11/2022]
Abstract
Bioprinting is an emerging approach for constructing sophisticated tissue analogues with detailed architectures such as vascular networks, which requires bioink fulfill the highly printable property and provide a cell-friendly microenvironment mimicking native extracellular matrix (ECM). Here, we developed a human placental ECM-derived bioink (hp-bioink) meeting the requirements of 3D printing for printability and bioactivity. We first decellularized the human placenta, followed by enzymatic digestion, dialysis, lyophilization, and re-solubilization to convert the extracts into hp-bioink. Then, we demonstrated that 3%-5% of hp-bioink can be printed with self-standing and 1%-2% of hp-bioink can be embedded with suspended hydrogels. Moreover, hp-bioink supports HUVEC assembly in vitro and angiogenesis in mice in vivo. Our research enriched the bank of human-derived bioink, and provided a new opportunity to further accelerate bioprinting research and application.
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Affiliation(s)
- Yongchao Duan
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Wenhui Huang
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Bo Zhan
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Yuanyuan Li
- Shanxi Provincial Peoples Hospital, No 29 Shuangtadong Street, Yinze district, Taiyuan, Taiyuan, Shanxi , 030012, CHINA
| | - Xue Xu
- Peking University People's Hospital, 11 Xizhimen South Street, Xicheng District, Beijing, Beijing, 100044, CHINA
| | - Kai Li
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Xia Li
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Xin Liu
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Shenglong Ding
- Beijing Tongren Hospital, 2 Chongwenmennei Dajie Dongcheng District, Beijing, Beijing, 100730, CHINA
| | - Shuo Wang
- Institute of Zoology Chinese Academy of Sciences, Institute of Zoology, Chinese Academy of Sciences, Chaoyang District, Beijing, 100101, CHINA
| | - Jia Guo
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Yukai Wang
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District, Beijing 100101, P.R.China, Chaoyang District, Beijing, 100101, CHINA
| | - Qi Gu
- Institute of Zoology Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang District District, Beijing, 100101, CHINA
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19
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Thant L, Kaku M, Kakihara Y, Mizukoshi M, Kitami M, Arai M, Kitami K, Kobayashi D, Yoshida Y, Maeda T, Saito I, Uoshima K, Saeki M. Extracellular Matrix-Oriented Proteomic Analysis of Periodontal Ligament Under Mechanical Stress. Front Physiol 2022; 13:899699. [PMID: 35669581 PMCID: PMC9163570 DOI: 10.3389/fphys.2022.899699] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2022] [Accepted: 04/11/2022] [Indexed: 11/22/2022] Open
Abstract
The periodontal ligament (PDL) is a specialized connective tissue that provides structural support to the tooth and is crucial for oral functions. The mechanical properties of the PDL are mainly derived from the tissue-specific composition and structural characteristics of the extracellular matrix (ECM). The ECM also plays key roles in determining cell fate in the cellular microenvironment thus crucial in the PDL tissue homeostasis. In the present study, we determined the comprehensive ECM profile of mouse molar PDL using laser microdissection and mass spectrometry-based proteomic analysis with ECM-oriented data curation. Additionally, we evaluated changes in the ECM proteome under mechanical loading using a mouse orthodontic tooth movement (OTM) model and analyzed potential regulatory networks using a bioinformatics approach. Proteomic changes were evaluated in reference to the novel second harmonic generation (SHG)-based fiber characterization. Our ECM-oriented proteomics approach succeeded in illustrating the comprehensive ECM profile of the mouse molar PDL. We revealed the presence of type II collagen in PDL, possibly associated with the load-bearing function upon occlusal force. Mechanical loading induced unique architectural changes in collagen fibers along with dynamic compositional changes in the matrisome profile, particularly involving ECM glycoproteins and matrisome-associated proteins. We identified several unique matrisome proteins which responded to the different modes of mechanical loading in PDL. Notably, the proportion of type VI collagen significantly increased at the mesial side, contributing to collagen fibrogenesis. On the other hand, type XII collagen increased at the PDL-cementum boundary of the distal side. Furthermore, a multifaceted bioinformatics approach illustrated the potential molecular cues, including PDGF signaling, that maintain ECM homeostasis under mechanical loading. Our findings provide fundamental insights into the molecular network underlying ECM homeostasis in PDL, which is vital for clinical diagnosis and development of biomimetic tissue-regeneration strategies.
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Affiliation(s)
- Lay Thant
- Division of Dental Pharmacology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
- Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
- Center for Advanced Oral Science, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Masaru Kaku
- Division of Bio-prosthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
- *Correspondence: Masaru Kaku,
| | - Yoshito Kakihara
- Division of Dental Pharmacology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Masaru Mizukoshi
- Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Megumi Kitami
- Division of Dental Pharmacology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
- Center for Advanced Oral Science, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Moe Arai
- Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Kohei Kitami
- Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Daiki Kobayashi
- Omics Unit, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Yutaka Yoshida
- Department of Structural Pathology, Kidney Research Center, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Takeyasu Maeda
- Center for Advanced Oral Science, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Isao Saito
- Division of Orthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Katsumi Uoshima
- Division of Bio-prosthodontics, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
| | - Makio Saeki
- Division of Dental Pharmacology, Faculty of Dentistry & Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan
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20
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Suda M, Shimizu I, Katsuumi G, Hsiao CL, Yoshida Y, Matsumoto N, Yoshida Y, Katayama A, Wada J, Seki M, Suzuki Y, Okuda S, Ozaki K, Nakanishi-Matsui M, Minamino T. Glycoprotein nonmetastatic melanoma protein B regulates lysosomal integrity and lifespan of senescent cells. Sci Rep 2022; 12:6522. [PMID: 35444208 PMCID: PMC9021310 DOI: 10.1038/s41598-022-10522-3] [Citation(s) in RCA: 42] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2021] [Accepted: 03/30/2022] [Indexed: 12/31/2022] Open
Abstract
Accumulation of senescent cells in various tissues has been reported to have a pathological role in age-associated diseases. Elimination of senescent cells (senolysis) was recently reported to reversibly improve pathological aging phenotypes without increasing rates of cancer. We previously identified glycoprotein nonmetastatic melanoma protein B (GPNMB) as a seno-antigen specifically expressed by senescent human vascular endothelial cells and demonstrated that vaccination against Gpnmb eliminated Gpnmb-positive senescent cells, leading to an improvement of age-associated pathologies in mice. The aim of this study was to elucidate whether GPNMB plays a role in senescent cells. We examined the potential role of GPNMB in senescent cells by testing the effects of GPNMB depletion and overexpression in vitro and in vivo. Depletion of GPNMB from human vascular endothelial cells shortened their replicative lifespan and increased the expression of negative cell cycle regulators. Conversely, GPNMB overexpression protected these cells against stress-induced premature senescence. Depletion of Gpnmb led to impairment of vascular function and enhanced atherogenesis in mice, whereas overexpression attenuated dietary vascular dysfunction and atherogenesis. GPNMB was upregulated by lysosomal stress associated with cellular senescence and was a crucial protective factor in maintaining lysosomal integrity. GPNMB is a seno-antigen that acts as a survival factor in senescent cells, suggesting that targeting seno-antigens such as GPNMB may be a novel strategy for senolytic treatments.
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Affiliation(s)
- Masayoshi Suda
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
| | - Ippei Shimizu
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
| | - Goro Katsuumi
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
- Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan
| | - Chieh Lun Hsiao
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
| | - Yohko Yoshida
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan
- Department of Advanced Senotherapeautics, Juntendo University Graduate School of Medicine, Tokyo, 113-8421, Japan
| | - Naomi Matsumoto
- Division of Biochemistry, School of Pharmacy, Iwate Medical University, Iwate, 028-3694, Japan
| | - Yutaka Yoshida
- Department of Structural Pathology, Kidney Research Center, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan
| | - Akihiro Katayama
- Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama, 700-8558, Japan
| | - Jun Wada
- Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama, 700-8558, Japan
| | - Masahide Seki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 277-8561, Japan
| | - Yutaka Suzuki
- Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, 277-8561, Japan
| | - Shujiro Okuda
- Division of Bioinformatics, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan
| | - Kazuyuki Ozaki
- Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan
| | - Mayumi Nakanishi-Matsui
- Division of Biochemistry, School of Pharmacy, Iwate Medical University, Iwate, 028-3694, Japan
| | - Tohru Minamino
- Department of Cardiovascular Biology and Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan.
- Department of Cardiovascular Biology and Medicine, Niigata University Graduate School of Medical and Dental Sciences, Niigata, 951-8510, Japan.
- Japan Agency for Medical Research and Development-Core Research for Evolutionary Medical Science and Technology (AMED-CREST), Japan Agency for Medical Research and Development, Tokyo, Japan.
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21
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Analysis of the Composition of Deinagkistrodon acutus Snake Venom Based on Proteomics, and Its Antithrombotic Activity and Toxicity Studies. Molecules 2022; 27:molecules27072229. [PMID: 35408629 PMCID: PMC9000436 DOI: 10.3390/molecules27072229] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Revised: 03/25/2022] [Accepted: 03/28/2022] [Indexed: 02/06/2023] Open
Abstract
There is a strong correlation between the composition of Deinagkistrodon acutus venom proteins and their potential pharmacological effects. The proteomic analysis revealed 103 proteins identified through label-free proteomics from 30 different snake venom families. Phospholipase A2 (30.0%), snaclec (21.0%), antithrombin (17.8%), thrombin (8.1%) and metalloproteinases (4.2%) were the most abundant proteins. The main toxicity of Deinagkistrodon acutus venom is hematotoxicity and neurotoxicity, and it acts on the lung. Deinagkistrodon acutus venom may have anticoagulant and antithrombotic effects. In summary, the protein profile and related toxicity and pharmacological activity of Deinagkistrodon acutus venom from southwest China were put forward for the first time. In addition, we revealed the relationship between the main toxicity, pharmacological effects, and the protein components of snake venom.
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22
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Feng S, Huang Q, Deng J, Jia W, Gong J, Xie D, Shen J, Liu L. DAB2IP suppresses tumor malignancy by inhibiting GRP75-driven p53 ubiquitination in colon cancer. Cancer Lett 2022; 532:215588. [PMID: 35150809 DOI: 10.1016/j.canlet.2022.215588] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/04/2021] [Revised: 02/03/2022] [Accepted: 02/03/2022] [Indexed: 02/07/2023]
Abstract
Increasing evidence has shown that DAB2IP acts as a tumor suppressor and plays an inhibitory role in many signals associated with tumorigenesis. However, the underlying mechanism of this function remains unclear. Our study shows that DAB2IP was positively associated with a good prognosis in patients with colorectal cancer and wild-type p53 expression. An in vitro assay showed that DAB2IP elicited potent tumor-suppressive effects by inhibiting cell invasiveness and colony formation and promoting cell apoptosis in wild-type p53 colon cancer cells. In addition, DAB2IP improved the stability of wild-type p53 by inhibiting its degradation in a ubiquitin-proteasome-dependent manner. Using mass spectrometry profiling, we revealed that DAB2IP and p53 interacted with the ubiquitin ligase-related protein GRP75. Mechanistically, DAB2IP is competitively bound to GRP75, thus reducing GRP75-driven p53 ubiquitination and degradation. Moreover, the Ras-GAP domain was required for the DAB2IP-GRP75 interaction and DAB2IP-mediated p53 ubiquitination. Finally, animal experiments revealed that DAB2IP inhibited tumor progression in vivo. In conclusion, our study presents a novel function of DAB2IP in GRP75-driven wild-type p53 degradation, providing new insight into DAB2IP-induced tumor suppression and a novel molecular interpretation of the p53 pathway.
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Affiliation(s)
- Shengjie Feng
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Qingwen Huang
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Jiao Deng
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Weiyi Jia
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Jianping Gong
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Daxing Xie
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China
| | - Jie Shen
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China.
| | - Liang Liu
- Department of Gastrointestinal Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Gastrointestinal Cancer Research Institute, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China.
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23
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Duan Y, Zhu W, Zhao X, Merzendorfer H, Chen J, Zou X, Yang Q. Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development. INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY 2022; 141:103718. [PMID: 34982980 DOI: 10.1016/j.ibmb.2021.103718] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Revised: 12/29/2021] [Accepted: 12/29/2021] [Indexed: 06/14/2023]
Abstract
Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, we demonstrate that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. Our findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein.
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Affiliation(s)
- Yanwei Duan
- School of Bioengineering, Dalian University of Technology, No. 2, Linggong Road, Dalian, 116024, China
| | - Weixing Zhu
- School of Bioengineering, Dalian University of Technology, No. 2, Linggong Road, Dalian, 116024, China
| | - Xiaoming Zhao
- Institute of Applied Biology, Shanxi University, Taiyuan, Shanxi, 030006, China
| | - Hans Merzendorfer
- Institute of Biology, University of Siegen, Adolf-Reichwein-Strasse 2, Siegen, 57068, Germany
| | - Jiqiang Chen
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Beijing, 100193, China
| | - Xu Zou
- State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Beijing, 100193, China
| | - Qing Yang
- School of Bioengineering, Dalian University of Technology, No. 2, Linggong Road, Dalian, 116024, China; State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Beijing, 100193, China; Shenzhen Branch, Guangdong Laboratory of Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, No 7 Pengfei Road, Shenzhen, 518120, China.
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24
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Zhao TC, Zhou ZH, Ju WT, Liang SY, Tang X, Zhu DW, Zhang ZY, Zhong LP. Mechanism of sensitivity to cisplatin, docetaxel, and 5-fluorouracil chemoagents and potential erbB2 alternatives in oral cancer with growth differentiation factor 15 overexpression. Cancer Sci 2021; 113:478-488. [PMID: 34826159 PMCID: PMC8819339 DOI: 10.1111/cas.15218] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 11/13/2021] [Accepted: 11/16/2021] [Indexed: 01/11/2023] Open
Abstract
The aim of this study was to: (a) explore the potential mechanism of cancer cell sensitivity to cisplatin, docetaxel, and 5‐fluorouracil (TPF) in oral squamous cell carcinoma (OSCC) patients overexpressing growth differentiation factor 15 (GDF15); and (b) identify potential alternative agents for patients who might not benefit from inductive TPF chemotherapy. The results indicated that OSCC cells overexpressing GDF15 were sensitive to TPF through a caspase‐9‐dependent pathway both in vitro and in vivo. Immunoprecipitation combined with mass spectrometry revealed that the erbB2 protein was a potential GDF15‐binding protein, which was verified by coimmunoprecipitation. Growth differentiation factor 15 overexpression promoted OSCC cell proliferation through erbB2 phosphorylation, as well as downstream AKT and Erk signaling pathways. When GDF15 expression was blocked, the phosphorylation of both the erbB2 and AKT/Erk pathways was downregulated. When OSCC cells with GDF15 overexpression were treated with the erbB2 phosphorylation inhibitor, CI‐1033, cell proliferation and xenograft growth colony formation were significantly blocked (P < .05). Thus, GDF15‐overexpressing OSCC tumors are sensitive to TPF chemoagents through caspase‐9‐dependent pathways. Growth differentiation factor 15 overexpression promotes OSCC proliferation through erbB2 phosphorylation. Thus, ErbB2 inhibitors could represent potential targeted drugs or an alternative therapy for OSCC patients with GDF15 overexpression.
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Affiliation(s)
- Tong-Chao Zhao
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Zhi-Hang Zhou
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Wu-Tong Ju
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Si-Yuan Liang
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Xiao Tang
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Dong-Wang Zhu
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Zhi-Yuan Zhang
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
| | - Lai-Ping Zhong
- Department of Oral and Maxillofacial-Head and Neck Oncology, College of Stomatology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.,National Clinical Research Center for Oral Diseases, Shanghai, China.,Shanghai Key Laboratory of Stomatology, Shanghai, China
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Pan X, Gao Q, Shen J, Xu T. 14-3-3 is a VP3-binding protein involved in Macrobrachium rosenbergii Taihu virus infection in shrimp. DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 2021; 122:104139. [PMID: 34023374 DOI: 10.1016/j.dci.2021.104139] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/23/2021] [Revised: 05/13/2021] [Accepted: 05/14/2021] [Indexed: 06/12/2023]
Abstract
Macrobrachium rosenbergii Taihu virus (MrTV) is a fierce pathogen that causes high mortality in M. rosenbergii larvae. Little is known about the pathogenesis of MrTV and host-virus interactions. In this study, a virus overlay protein binding assay (VOPBA), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was carried out to search for novel host molecules that bind with VP3, one of the main capsid proteins of MrTV. Macrobrachium rosenbergii 14-3-3 protein (Mr14-3-3) was identified as the binding protein of VP3, which was further confirmed by co-immunoprecipitation (Co-IP) and co-localization assay. A preincubation assay was developed, which indicated that preincubation with recombinant Mr14-3-3 (rMr14-3-3) could significantly decrease the expression level of VP3 in MrTV-infected M. rosenbergii larvae, suggesting that preincubation with rMr14-3-3 could partially block MrTV infection. This study revealed that Mr14-3-3 acts as a binding protein for MrTV-VP3 and plays an important role in MrTV infection, offering a potential target for the development of anti-MrTV therapies.
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Affiliation(s)
- Xiaoyi Pan
- Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China
| | - Qiang Gao
- Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China
| | - Jinyu Shen
- Zhejiang Institute of Freshwater Fisheries, Huzhou, 313001, China
| | - Ting Xu
- School of Life Sciences, Shaoxing University, Shaoxing, 312000, China.
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Identification of a specific surface epitope of OmpC for Escherichia coli O157:H7 with protein topology facilitated affinity mass spectrometry. Appl Microbiol Biotechnol 2021; 105:6819-6833. [PMID: 34432131 PMCID: PMC8426304 DOI: 10.1007/s00253-021-11511-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2021] [Revised: 08/04/2021] [Accepted: 08/07/2021] [Indexed: 11/03/2022]
Abstract
Abstract
The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Key points • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC–MS/MS. • The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11511-8.
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Deletion of Yersinia pestis ail causes temperature sensitive pleiotropic effects including cell lysis that are suppressed by carbon source, cations, or loss of phospholipase A activity. J Bacteriol 2021; 203:e0036121. [PMID: 34398663 PMCID: PMC8508112 DOI: 10.1128/jb.00361-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Maintenance of phospholipid (PL) and lipopoly- or lipooligo-saccharide (LPS or LOS) asymmetry in the outer membrane (OM) of Gram-negative bacteria is essential but poorly understood. The Yersinia pestis OM Ail protein was required to maintain lipid homeostasis and cell integrity at elevated temperature (37° C). Loss of this protein had pleiotropic effects. A Y. pestis Δail mutant and KIM6+ wild- type were systematically compared for (i) growth requirements at 37° C, (ii) cell structure, (iii) antibiotic and detergent sensitivity, (iv) proteins released into supernates, (v) induction of the heat shock response, and (vi) physiological and genetic suppressors that restored the wild- type phenotype. The Δail mutant grew normally at 28° C but lysed at 37° C when it entered stationary phase as shown by cell count, SDS-PAGE of cell supernatants, and electron microscopy. Immuno-fluorescent microscopy showed that the Δail mutant did not assemble Caf1 capsule. Expression of heat shock promoters rpoE or rpoH fused to a lux operon reporter were not induced when the Δail mutant was shifted from the 28° C to 37° C (p<0.001 and p<0.01 respectively). Mutant lysis was suppressed by addition of 11 mM glucose, 22 or 44 mM glycerol, 2.5 mM Ca2+, or 2.5 mM Mg2+ to the growth medium, or by a mutation in the phospholipase A gene (pldA::miniTn5, ΔpldA, or PldAS164A). A model, accounting for the temperature-sensitive lysis of the Δail mutant and the Ail-dependent stabilization of the OM tetraacylated LOS at 37°C is presented. IMPORTANCE The Gram-negative pathogen, Yersinia pestis, transitions between a flea vector (ambient temperature) and a mammalian host (37° C). In response to 37° C, Y. pestis modifies its outer membrane (OM) by reducing the fatty acid content in lipid A, changing the outer leaflet from being predominantly hexaacylated to being predominantly tetraacylated. It also increases the Ail concentration, so it becomes the most prominent OM protein. Both measures are needed for Y. pestis to evade the host innate immune response. Deletion of ail destabilizes the OM at 37° C causing the cells to lyse. These results show that a protein is essential for maintaining lipid asymmetry and lipid homeostasis in the bacterial OM.
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Abstract
Here, we describe a protocol for a photoaffinity labeling probe strategy for target deconvolution in live cells. We made a chemical probe by incorporation of a photoreactive group to covalently cross-link with adjacent amino acid residues upon UV irradiation. Click chemistry-based enrichment captures labeled proteins for proteomic analysis. Here, we detail specifics for finding targets of LXRβ, but the protocol has potential for application to other targets. For complete details on the use and execution of this protocol, please refer to Seneviratne et al. (2020).
Protocol detailing photoaffinity probe strategy for target deconvolution in live cells Competition with parent compound demonstrates specific binding Photoaffinity label provides evidence of small-molecule binding to LXRβ Click chemistry-based enrichment captures labeled proteins for proteomic analysis
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29
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Chen MX, Cheng S, Lei L, Zhang XF, Liu Q, Lin A, Wallis CU, Lukowicz MJ, Sham PC, Li Q, Ao LJ. The effects of maternal SSRI exposure on the serotonin system, prefrontal protein expression and behavioral development in male and female offspring rats. Neurochem Int 2021; 146:105041. [PMID: 33836218 DOI: 10.1016/j.neuint.2021.105041] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/03/2020] [Revised: 03/21/2021] [Accepted: 03/31/2021] [Indexed: 11/24/2022]
Abstract
Fluoxetine (FLX), a commonly used selective serotonin reuptake inhibitor, is often used to treat depression during pregnancy. However, prenatal exposure to FLX has been associated with a series of neuropsychiatric illnesses. The use of a rodent model can provide a clear indication as to whether prenatal exposure to SSRIs, independent of maternal psychiatric disorders or genetic syndromes, can cause long-term behavioral abnormalities in offspring. Thus, the present study aimed to explore whether prenatal FLX exposure causes long-term neurobehavioral effects, and identify the underlying mechanism between FLX and abnormal behaviors. In our study, 12/mg/kg/day of FLX or equal normal saline (NS) was administered to pregnant Sprague-Dawley (SD) rats (FLX = 30, NS = 27) on gestation day 11 till birth. We assessed the physical development and behavior of offspring, and in vivo magnetic resonance spectroscopy (MRS) was conducted to quantify biochemical alterations in the prefrontal cortex (PFC). Ex vivo measurements of brain serotonin level and a proteomic analysis were also undertaken. Our results showed that the offspring (male offspring in particular) of fluoxetine exposed mothers showed delayed physical development, increased anxiety-like behavior, and impaired social interaction. Moreover, down-regulation of 5-HT and SERT expression were identified in the PFC. We also found that prenatal FLX exposure significantly decreased NAA/tCr with 1H-MRS in the PFC of offspring. Finally, a proteomic study revealed sex-dependent differential protein expression. These findings may have translational importance suggesting that using SSRI medication alone in pregnant mothers may result in developmental delay in their offspring. Our results also help guide the choice of outcome measures in identifying of molecular and developmental mechanisms.
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Affiliation(s)
- Mo Xian Chen
- School of Rehabilitation, Kunming Medical University, Kunming, China
| | - Shu Cheng
- Department of Rehabilitation, China Resources & WISCO General Hospital, Wuhan, China
| | - Lei Lei
- Rehabilitation Medicine Department, The Affiliated Hospital of Southwest Medical University, Tai Ping Road, Luzhou, Sichuan, China
| | - Xiao Fan Zhang
- Department of Psychiatry, Tongji Hospital of Huazhong University of Science and Technology (HUST), China
| | - Qiang Liu
- Department of Surgery, The Chinese University of Hong Kong, Hong Kong, SAR, China
| | - Aijin Lin
- School of Rehabilitation, Kunming Medical University, Kunming, China
| | | | | | - Pak C Sham
- Department of Psychiatry, The University of Hong Kong, Hong Kong, SAR, China; State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong, SAR, China; Centre for Genomic Sciences, The University of Hong Kong, Hong Kong, SAR, China
| | - Qi Li
- Department of Psychiatry, The University of Hong Kong, Hong Kong, SAR, China; State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Hong Kong, SAR, China.
| | - Li Juan Ao
- School of Rehabilitation, Kunming Medical University, Kunming, China.
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30
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Yan Q, Zhao L, Wang W, Pi X, Han G, Wang J, Cheng L, He YK, Kuang T, Qin X, Sui SF, Shen JR. Antenna arrangement and energy-transfer pathways of PSI-LHCI from the moss Physcomitrella patens. Cell Discov 2021; 7:10. [PMID: 33589616 PMCID: PMC7884438 DOI: 10.1038/s41421-021-00242-9] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2020] [Accepted: 12/21/2020] [Indexed: 02/07/2023] Open
Abstract
Plants harvest light energy utilized for photosynthesis by light-harvesting complex I and II (LHCI and LHCII) surrounding photosystem I and II (PSI and PSII), respectively. During the evolution of green plants, moss is at an evolutionarily intermediate position from aquatic photosynthetic organisms to land plants, being the first photosynthetic organisms that landed. Here, we report the structure of the PSI–LHCI supercomplex from the moss Physcomitrella patens (Pp) at 3.23 Å resolution solved by cryo-electron microscopy. Our structure revealed that four Lhca subunits are associated with the PSI core in an order of Lhca1–Lhca5–Lhca2–Lhca3. This number is much decreased from 8 to 10, the number of subunits in most green algal PSI–LHCI, but the same as those of land plants. Although Pp PSI–LHCI has a similar structure as PSI–LHCI of land plants, it has Lhca5, instead of Lhca4, in the second position of Lhca, and several differences were found in the arrangement of chlorophylls among green algal, moss, and land plant PSI–LHCI. One chlorophyll, PsaF–Chl 305, which is found in the moss PSI–LHCI, is located at the gap region between the two middle Lhca subunits and the PSI core, and therefore may make the excitation energy transfer from LHCI to the core more efficient than that of land plants. On the other hand, energy-transfer paths at the two side Lhca subunits are relatively conserved. These results provide a structural basis for unravelling the mechanisms of light-energy harvesting and transfer in the moss PSI–LHCI, as well as important clues on the changes of PSI–LHCI after landing.
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Affiliation(s)
- Qiujing Yan
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Liang Zhao
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, China
| | - Wenda Wang
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
| | - Xiong Pi
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, China
| | - Guangye Han
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
| | - Jie Wang
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China.,University of Chinese Academy of Sciences, Beijing, 100049, China
| | - Lingpeng Cheng
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, China
| | - Yi-Kun He
- College of Life Sciences, Department of Chemistry, Capital Normal University, Beijing, 100048, China
| | - Tingyun Kuang
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China
| | - Xiaochun Qin
- School of Biological Science and Technology, University of Jinan, Jinan, Shandong, 250022, China.
| | - Sen-Fang Sui
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology & Frontier Research Center for Biological Structure, School of Life Sciences, Tsinghua University, Beijing, 100084, China. .,Department of Biology, Southern University of Science and Technology, Shenzhen, Guangdong, 518055, China.
| | - Jian-Ren Shen
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, China. .,Research Institute for Interdisciplinary Science, and Graduate School of Natural Science and Technology, Okayama University, Okayama, 700-8530, Japan.
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31
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Efficient production of the anti-aging drug Cycloastragenol: insight from two Glycosidases by enzyme mining. Appl Microbiol Biotechnol 2020; 104:9991-10004. [PMID: 33119795 DOI: 10.1007/s00253-020-10966-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2020] [Revised: 10/09/2020] [Accepted: 10/18/2020] [Indexed: 10/23/2022]
Abstract
The telomerase activator cycloastragenol (CA) is regarded as a potential anti-aging drug with promising applications in the food and medical industry. However, one remaining challenge is the low efficiency of CA production. Herein, we developed an enzyme-based approach by applying two enzymes (β-xylosidase: Xyl-T; β-glucosidase: Bgcm) for efficient CA production. Both key glycosidases, mined by activity tracking or homology sequence screening, were successfully over-expressed and showed prominent enzymatic activity profiles, including widely pH stability (Xyl-T: pH 3.0-8.0; Bgcm: pH 4.0-10.0), high catalytic efficiency (kcat/Km: 0.096 mM-1s-1 (Xyl-T) and 3.08 mM-1s-1 (Bgcm)), and mesophilic optimum catalytic temperature (50 °C). Besides, the putative catalytic residues (Xyl-T: Asp311/Glu 521; Bgcm: Asp311/Glu 521) and the potential substrate-binding mechanism of Xyl-T and Bgcm were predicted by comprehensive computational analysis, providing valuable insight into the hydrolysis of substrates at the molecular level. Notably, a rationally designed two-step reaction process was introduced to improve the CA yield and increased up to 96.5% in the gram-scale production, providing a potential alternative for the industrial CA bio-production. In essence, the explored enzymes, the developed enzyme-based approach, and the obtained knowledge from catalytic mechanisms empower researchers to further engineer the CA production and might be applied for other chemicals synthesis. KEY POINTS: • A β-xylosidase and a β-glucosidase were mined to hydrolyze ASI into CA. • The two recombinant glycosidases showed prominent catalytic profiles. • Two-step enzymatic catalysis for CA production from ASI was developed. Graphical abstract.
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Guo L, Wang J, Gou Y, Tan L, Liu H, Pan Y, Zhao Y. Comparative proteomics reveals stress responses of Vibrio parahaemolyticus biofilm on different surfaces: Internal adaptation and external adjustment. THE SCIENCE OF THE TOTAL ENVIRONMENT 2020; 731:138386. [PMID: 32417469 DOI: 10.1016/j.scitotenv.2020.138386] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/07/2020] [Revised: 03/27/2020] [Accepted: 03/31/2020] [Indexed: 06/11/2023]
Abstract
Vibrio parahaemolyticus is a kind of gram-negative marine pathogen, which usually adheres to stainless steel (SS), glass (GS) and other abiotic surfaces in aquaculture and food processing in the form of biofilm and causes the spread of gastrointestinal illness. However, the deeply survival adaptation mechanism of V. parahaemolyticus biofilm cells on these contact surface remained unclear. Here, proteomics was used to investigated the physiological response of the V. parahaemolyticus biofilms cells to different abiotic surfaces (SS, GS and polystyrene (PS)). In addition, the effect of contact materials on the physical-chemical properties of biofilms are also characterized. Results showed that the expression of proteins of biofilm cells established on the SS surface were mainly related to the alleviation of metal ion stress and toxicity. The up-regulated proteins in the biofilm cells formed on the GS surface were mainly involved in the biological processes of sugar uptake, protein synthesis and bacterial chemotaxis. Meanwhile, the significantly expressed proteins in the biofilm cells formed on the PS surface were mainly involved in the cellular physiological activity of aromatic compound metabolism, osmotic stress and nutrient transport. All functional proteins mentioned above were closely related to the interaction characteristics of the contact surface and biofilm. This study provided an in-depth comparison of V. parahaemolyticus biofilm formation on these three abiotic surfaces, and presented a model in first time for the adaptation behavior of biofilm cells on different surfaces as affected by metal ion stress, nutrition, osmotic stress, and sugar utilization, which could facilitate an efficient control strategy for biofilm formation in industrial field.
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Affiliation(s)
- Linxia Guo
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China
| | - Jingjing Wang
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China; Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China; Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China; Department of Food Science, Foshan University, Foshan, 528000, China
| | - Yi Gou
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China
| | - Ling Tan
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China
| | - Haiquan Liu
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China; Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China; Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China; Engineering Research Center of Food Thermal-processing Technology, Shanghai Ocean University, Shanghai 201306, China
| | - Yingjie Pan
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China; Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China; Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China
| | - Yong Zhao
- College of Food Science & Technology, Shanghai Ocean University, Shanghai, China; Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture, Shanghai, China; Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai, China.
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33
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Mei X, Chang Y, Shen J, Zhang Y, Xue C. Expression and characterization of a novel alginate-binding protein: A promising tool for investigating alginate. Carbohydr Polym 2020; 246:116645. [PMID: 32747278 DOI: 10.1016/j.carbpol.2020.116645] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2020] [Revised: 05/06/2020] [Accepted: 06/11/2020] [Indexed: 12/26/2022]
Abstract
Alginate is a commercially important polysaccharide widely applied in various industries. Carbohydrate-binding proteins could be utilized as desirable tools in the investigation and further applications of polysaccharides. Few alginate-binding proteins have hitherto been characterized and reported. In the present study, a novel alginate-binding protein ABP_Wf, consisting of two "orphan" carbohydrate-binding modules, was cloned from a predicted alginate utilization locus of marine bacterium Wenyingzhuangia funcanilytica, and expressed in Escherichia coli. ABP_Wf exhibited a specific binding capacity to alginate, and the association constant (Ka) and affinity (KD) were 1.94 × 103 M-1s-1 and 1.16 × 10-6 M. It was confirmed that the binding capacity of ABP_Wf to alginate is attributed to its constituent CBM16 domain rather than the CBM44 domain. The potentials of ABP_Wf in the semi-quantitative detection and the in situ visualization of alginate were evaluated, which implied that ABP_Wf could be served as a promising tool for investigating alginate.
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Affiliation(s)
- Xuanwei Mei
- College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China
| | - Yaoguang Chang
- College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China.
| | - Jingjing Shen
- College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China
| | - Yuying Zhang
- College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China
| | - Changhu Xue
- College of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao, 266003, China; Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao, 266237, China
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34
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Xing CY, Fan YC, Chen X, Guo JS, Shen Y, Yan P, Fang F, Chen YP. A self-assembled nanocompartment in anammox bacteria for resisting intracelluar hydroxylamine stress. THE SCIENCE OF THE TOTAL ENVIRONMENT 2020; 717:137030. [PMID: 32062250 DOI: 10.1016/j.scitotenv.2020.137030] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/04/2019] [Revised: 01/29/2020] [Accepted: 01/29/2020] [Indexed: 06/10/2023]
Abstract
Anammox bacteria play an important role in the global nitrogen cycle, but research on anammoxosome structure is still in its initial stages. In particular, the anammox bacteria genome contains nanocompartments gene loci. However, the function and structure of the nanocompartments in anammox bacteria is poorly understood. We apply genetic engineering to demonstrate the self-assembled nanocompartments of anammox bacteria. The encapsulin shell protein (cEnc) and cargo protein hydroxylamine oxidoreductase (HAO) can self-assemble to form regular nanocompartments (about 128 nm in diameter) in vitro. Cell growth curve tests show that nanocompartments help model bacteria resist hydroxylamine (NH2OH) stress. Batch test results and transcriptional data show that cEnc and HAO are highly expressed in response to the negative effects of NH2OH on anammox efficiency, predicting a potential role of nanocompartments in helping anammox bacteria resist NH2OH stress. These findings improve our understanding of the mechanisms by which anammox bacteria respond to harmful environmental metabolites.
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Affiliation(s)
- Chong-Yang Xing
- Key Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green and Intelligence Technology, Chinese Academy of Sciences, Chongqing 400714, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yu-Chen Fan
- Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China
| | - Xuan Chen
- Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China
| | - Jin-Song Guo
- Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China
| | - Yu Shen
- National Base of International Science and Technology Cooperation for Intelligent Manufacturing Service, Chongqing Technology and Business University, Chongqing 400067, China
| | - Peng Yan
- Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China
| | - Fang Fang
- Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China
| | - You-Peng Chen
- Key Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green and Intelligence Technology, Chinese Academy of Sciences, Chongqing 400714, China; University of Chinese Academy of Sciences, Beijing 100049, China; Key Laboratory of the Three Gorges Reservoir Region's Eco-Environments of MOE, Chongqing University, Chongqing 400045, China.
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35
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Hu K, Wang X, Zhu J, Liu A, Ao X, He L, Chen S, Zhou K, Yang Y, Zou L, Liu S. Characterization of carbaryl-degrading strain Bacillus licheniformis B-1 and its hydrolase identification. Biodegradation 2020; 31:139-152. [PMID: 32306137 DOI: 10.1007/s10532-020-09899-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2020] [Accepted: 04/08/2020] [Indexed: 10/24/2022]
Abstract
Pesticides introduced inadvertently or deliberately into environment by anthropogenic activity have caused growing global public concern, therefore the search of approaches for elimination of such xenobiotics should be encouraged. A cypermethrin-degrading bacterial strain Bacillus licheniformis B-1 was found to efficiently degrade carbaryl in LB medium at concentrations of 50-300 mg L-1 within 48 h, during which temperature and pH played important roles as reflected by increase in pollutant depletion. A stimulatory effect of Fe3+ and Mn2+ on microbial growth was observed, whereas Cu2+ caused inhibition of degradation. Results showed that 1-naphthol was a major transformation product of carbaryl which was further metabolised. An approximately 29 kDa carbaryl-degrading enzyme was purified from B-1 with 15.93-fold purification and an overall yield of 6.02% was achieved using ammonium sulphate precipitation, DEAE-Sepharose CL-6B anion-exchange chromatography and Sephadex G-100 gel filtration. The enzyme was identified through nano reversed-phase liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry as a phosphodiesterase (PDE). This is the first report on the characterization of carbaryl-degrading by Bacillus spp. and the role of a PDE in carbaryl-detoxifying. Also, strain B-1 showed versatile in carbosulfan, isoprocarb and chlorpyrifos degradation, demonstrating as ideal candidate for environment bioremediation.
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Affiliation(s)
- Kaidi Hu
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China.,Departament d'Enginyeria Química, Biològica i Ambiental, Escola d'Enginyeria, Universitat Autònoma de Barcelona, Bellaterra, Barcelona, 08193, Spain
| | - Xingjie Wang
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Jiawen Zhu
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Aiping Liu
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Xiaolin Ao
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China.,Institute of Food Processing and Safety, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Li He
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Shujuan Chen
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Kang Zhou
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Yong Yang
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China
| | - Likou Zou
- College of Resources, Sichuan Agricultural University, Wenjiang, Sichuan, 611130, People's Republic of China
| | - Shuliang Liu
- College of Food Science, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China. .,Institute of Food Processing and Safety, Sichuan Agricultural University, Ya'an, Sichuan, 625014, People's Republic of China.
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36
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Qin G, Wang X, Ye S, Li Y, Chen M, Wang S, Qin T, Zhang C, Li Y, Long Q, Hu H, Shi D, Li J, Zhang K, Zhai Q, Tang Y, Kang T, Lan P, Xie F, Lu J, Deng W. NPM1 upregulates the transcription of PD-L1 and suppresses T cell activity in triple-negative breast cancer. Nat Commun 2020; 11:1669. [PMID: 32245950 PMCID: PMC7125142 DOI: 10.1038/s41467-020-15364-z] [Citation(s) in RCA: 119] [Impact Index Per Article: 23.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2018] [Accepted: 02/28/2020] [Indexed: 12/31/2022] Open
Abstract
Programmed cell death protein-1 (PD-1)/programmed cell death ligand-1 (PD-L1) interaction plays a crucial role in tumor-associated immune escape. Here, we verify that triple-negative breast cancer (TNBC) has higher PD-L1 expression than other subtypes. We then discover that nucleophosmin (NPM1) binds to PD-L1 promoter specifically in TNBC cells and activates PD-L1 transcription, thus inhibiting T cell activity in vitro and in vivo. Furthermore, we demonstrate that PARP1 suppresses PD-L1 transcription through its interaction with the nucleic acid binding domain of NPM1, which is required for the binding of NPM1 at PD-L1 promoter. Consistently, the PARP1 inhibitor olaparib elevates PD-L1 expression in TNBC and exerts a better effect with anti-PD-L1 therapy. Together, our research has revealed NPM1 as a transcription regulator of PD-L1 in TNBC, which could lead to potential therapeutic strategies to enhance the efficacy of cancer immunotherapy.
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MESH Headings
- Adult
- Aged
- Animals
- Antineoplastic Agents, Immunological/pharmacology
- Antineoplastic Agents, Immunological/therapeutic use
- Antineoplastic Combined Chemotherapy Protocols/pharmacology
- Antineoplastic Combined Chemotherapy Protocols/therapeutic use
- B7-H1 Antigen/antagonists & inhibitors
- B7-H1 Antigen/genetics
- B7-H1 Antigen/metabolism
- Breast/pathology
- Cell Line, Tumor
- DNA-Binding Proteins
- Disease Models, Animal
- Drug Synergism
- Female
- Gene Expression Regulation, Neoplastic/drug effects
- Gene Expression Regulation, Neoplastic/immunology
- Gene Knockdown Techniques
- Humans
- Kaplan-Meier Estimate
- Lymphocytes, Tumor-Infiltrating/drug effects
- Lymphocytes, Tumor-Infiltrating/immunology
- Lymphocytes, Tumor-Infiltrating/metabolism
- Mice
- Middle Aged
- Nuclear Proteins/genetics
- Nuclear Proteins/metabolism
- Nucleophosmin
- Phthalazines/pharmacology
- Phthalazines/therapeutic use
- Piperazines/pharmacology
- Piperazines/therapeutic use
- Poly (ADP-Ribose) Polymerase-1/antagonists & inhibitors
- Poly (ADP-Ribose) Polymerase-1/metabolism
- Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
- Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use
- Prognosis
- Promoter Regions, Genetic/genetics
- T-Lymphocytes/drug effects
- T-Lymphocytes/immunology
- T-Lymphocytes/metabolism
- Tissue Array Analysis
- Transcriptional Activation/immunology
- Triple Negative Breast Neoplasms/drug therapy
- Triple Negative Breast Neoplasms/genetics
- Triple Negative Breast Neoplasms/immunology
- Triple Negative Breast Neoplasms/mortality
- Up-Regulation/drug effects
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Affiliation(s)
- Ge Qin
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
- The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Xin Wang
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Shubiao Ye
- The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Yizhuo Li
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Miao Chen
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Shusen Wang
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Tao Qin
- Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China
| | - Changlin Zhang
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Yixin Li
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Qian Long
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Huabin Hu
- The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Dingbo Shi
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Jiaping Li
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Kai Zhang
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Qinglian Zhai
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Yanlai Tang
- The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Tiebang Kang
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Ping Lan
- The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Fangyun Xie
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China
| | - Jianjun Lu
- The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Wuguo Deng
- Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou, China.
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Sun H, Shang M, Tang Z, Jiang H, Dong H, Zhou X, Lin Z, Shi C, Ren P, Zhao L, Shi M, Zhou L, Pan H, Chang O, Li X, Huang Y, Yu X. Oral delivery of Bacillus subtilis spores expressing Clonorchis sinensis paramyosin protects grass carp from cercaria infection. Appl Microbiol Biotechnol 2020; 104:1633-1646. [PMID: 31912200 PMCID: PMC7223688 DOI: 10.1007/s00253-019-10316-0] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2019] [Revised: 12/02/2019] [Accepted: 12/11/2019] [Indexed: 02/07/2023]
Abstract
Clonorchis sinensis (C. sinensis), an important fishborne zoonotic parasite threatening public health, is of major socioeconomic importance in epidemic areas. Effective strategies are still urgently expected to prevent against C. sinensis infection. In the present study, paramyosin of C. sinensis (CsPmy) was stably and abundantly expressed on the surface of Bacillus subtilis spores. The recombinant spores (B.s-CotC-CsPmy) were incorporated in the basal pellets diet in three different dosages (1 × 105, 1 × 108, 1 × 1011 CFU/g pellets) and orally administrated to grass carp (Ctenopharyngodon idella). The immune responses and intestinal microbiota in the treated grass carp were investigated. Results showed that specific anti-CsPmy IgM levels in sera, skin mucus, bile, and intestinal mucus, as well as mRNA levels of IgM and IgZ in the spleen and head kidney, were significantly increased in B.s-CotC-CsPmy-1011 group. Besides, transcripts levels of IL-8 and TNF-αin the spleen and head kidney were also significantly elevated than the control groups. Moreover, mRNA levels of tight junction proteins in the intestines of B.s-CotC-CsPmy-1011 group increased. Potential pathogenetic bacteria with lower abundance and higher abundances of candidate probiotics and bacteria associated with digestion in 1 × 1011 CFU/g B.s-CotC-CsPmy spores administrated fishes could be detected compared with control group. The amount of metacercaria in per gram fish flesh was statistically decreased in 1 × 1011 CFU/g B.s-CotC-CsPmy spores orally immunized group. Our work demonstrated that B. subtilis spores presenting CsPmy on the surface could be a promising effective, safe, and needle-free candidate vaccine against C. sinensis infection for grass carp.
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Affiliation(s)
- Hengchang Sun
- Department of Laboratory Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Mei Shang
- Department of Laboratory Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Zeli Tang
- Department of Cell Biology and Genetics, School of Pre-clinical Medicine, Guangxi Medical University, Nanning, 530021, China
| | - Hongye Jiang
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Huimin Dong
- Department of Laboratory Medicine, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Xinyi Zhou
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Zhipeng Lin
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Cunbin Shi
- Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River, Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China
| | - Pengli Ren
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Lu Zhao
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Mengchen Shi
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Lina Zhou
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Houjun Pan
- Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River, Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China
| | - Ouqin Chang
- Key Laboratory of Fishery Drug Development, Ministry of Agriculture, Pearl River, Fisheries Research Institute, Chinese Academy of Fishery Sciences, Guangzhou, Guangdong, China
| | - Xuerong Li
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China
| | - Yan Huang
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China.
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China.
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China.
| | - Xinbing Yu
- Department of parasitology, Zhongshan School of medicine, Sun Yat-sen University, Guangzhou, China.
- Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education,, Guangzhou, Guangdong, China.
- Provincial Engineering Technology Research Center for Biological Vector Control, Guangzhou, 510080, Guangdong, China.
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Li W, Hu L, Xie Z, Xu H, Li M, Cui T, He ZG. Cyclic di-GMP integrates functionally divergent transcription factors into a regulation pathway for antioxidant defense. Nucleic Acids Res 2019; 46:7270-7283. [PMID: 29982829 PMCID: PMC6101608 DOI: 10.1093/nar/gky611] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/13/2018] [Accepted: 06/27/2018] [Indexed: 11/14/2022] Open
Abstract
Cyclic diguanylate monophosphate (c-di-GMP) is a global signaling molecule that modulates diverse cellular processes through its downstream receptors. However, no study has fully clarified the mechanisms by which c-di-GMP organizes functionally divergent regulators to drive the gene expression for coping with environmental stress. Here, we reported that c-di-GMP can integrate two functionally opposite receptor transcription factors, namely, LtmA and HpoR, into a pathway to regulate the antioxidant processes in Mycobacterium smegmatis. In contrast to HpoR, LtmA is an activator that positively regulates the expression of redox gene clusters and the mycobacterial H2O2 resistance. LtmA can physically interact with HpoR. A high level of c-di-GMP stimulates the positive regulation of LtmA and boosts the physical interaction between the two regulators, further enhancing the DNA-binding ability of LtmA and reducing the inhibitory activity of HpoR. Therefore, upon exposure to oxidative stress, c-di-GMP can orchestrate functionally divergent transcription factors to trigger antioxidant defense in mycobacteria. This finding presents a noteworthy example of how a bacterium remodels its transcriptional network via c-di-GMP in response to environmental stress.
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Affiliation(s)
- Weihui Li
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Lihua Hu
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Zhiwei Xie
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Hui Xu
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Meng Li
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Tao Cui
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
| | - Zheng-Guo He
- National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China
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Cheng L, Zhang S, Yang L, Wang Y, Yu B, Zhang F. Comparative proteomics illustrates the complexity of Fe, Mn and Zn deficiency-responsive mechanisms of potato (Solanum tuberosum L.) plants in vitro. PLANTA 2019; 250:199-217. [PMID: 30976909 DOI: 10.1007/s00425-019-03163-w] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/17/2018] [Accepted: 04/05/2019] [Indexed: 05/05/2023]
Abstract
The present study is the first to integrate physiological and proteomic data providing information on Fe, Mn and Zn deficiency-responsive mechanisms of potato plants in vitro. Micronutrient deficiency is an important limiting factor for potato production that causes substantial tuber yield and quality losses. To under the underlying molecular mechanisms of potato in response to Fe, Mn and Zn deficiency, a comparative proteomic approach was applied. Leaf proteome change of in vitro-propagated potato plantlets subjected to a range of Fe-deficiency treatments (20, 10 and 0 μM Na-Fe-EDTA), Mn-deficiency treatments (1 and 0 μM MnCl2·4H2O) and Zn-deficiency treatment (0 μM ZnCl2) using two-dimensional gel electrophoresis was analyzed. Quantitative image analysis showed a total of 146, 55 and 42 protein spots under Fe, Mn and Zn deficiency with their abundance significantly altered (P < 0.05) more than twofold, respectively. By MALDI-TOF/TOF MS analyses, the differentially abundant proteins were found mainly involved in bioenergy and metabolism, photosynthesis, defence, redox homeostasis and protein biosynthesis/degradation under the metal deficiencies. Signaling, transport, cellular structure and transcription-related proteins were also identified. The hierarchical clustering results revealed that these proteins were involved in a dynamic network in response to Fe, Mn and Zn deficiency. All these metal deficiencies caused cellular metabolic remodeling to improve metal acquisition and distribution in potato plants. The reduced photosynthetic efficiency occurred under each metal deficiency, yet Fe-deficient plants showed a more severe damage of photosynthesis. More defence mechanisms were induced by Fe deficiency than Mn and Zn deficiency, and the antioxidant systems showed different responses to each metal deficiency. Reprogramming of protein biosynthesis/degradation and assembly was more strongly required for acclimation to Fe deficiency. The signaling cascades involving auxin and NDPKs might also play roles in micronutrient stress signaling and pinpoint interesting candidates for future studies. Our results first provide an insight into the complex functional and regulatory networks in potato plants under Fe, Mn and Zn deficiency.
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Affiliation(s)
- Lixiang Cheng
- College of Agronomy, Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Gansu Agricultural University, Lanzhou, 730070, China
| | - Shaomei Zhang
- College of Agronomy, Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Gansu Agricultural University, Lanzhou, 730070, China
| | - Lili Yang
- College of Agronomy, Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Gansu Agricultural University, Lanzhou, 730070, China
| | - Yuping Wang
- College of Horticulture, Gansu Agricultural University, Lanzhou, China
| | - Bin Yu
- College of Agronomy, Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Gansu Agricultural University, Lanzhou, 730070, China
| | - Feng Zhang
- College of Agronomy, Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Key Laboratory of Crop Improvement and Germplasm Enhancement, Gansu Agricultural University, Lanzhou, 730070, China.
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40
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Xu W, Deng B, Lin P, Liu C, Li B, Huang Q, Zhou H, Yang J, Qu L. Ribosome profiling analysis identified a KRAS-interacting microprotein that represses oncogenic signaling in hepatocellular carcinoma cells. SCIENCE CHINA-LIFE SCIENCES 2019; 63:529-542. [DOI: 10.1007/s11427-019-9580-5] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/16/2019] [Accepted: 05/28/2019] [Indexed: 12/13/2022]
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Yao Q, Yu Z, Liu P, Zheng H, Xu Y, Sai S, Wu Y, Zheng C. High Efficient Expression and Purification of Human Epidermal Growth Factor in Arachis Hypogaea L. Int J Mol Sci 2019; 20:ijms20082045. [PMID: 31027239 PMCID: PMC6515032 DOI: 10.3390/ijms20082045] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2019] [Revised: 04/21/2019] [Accepted: 04/24/2019] [Indexed: 12/04/2022] Open
Abstract
Background: Human epidermal growth factor (hEGF) has drawn intense research attention due to its potential ability to promote healing of serious injuries, such as cuts, burns, and diabetic ulcers. Although hEGF displays prospective clinical value, the growth factor is restricted to the treatment of chronic diabetic ulcers because of its high production cost. Methods: Leguminous plant peanut (Arachis hypogaea L.) hairy roots contain relatively few toxic and harmful substances, and tested as an excellent production system for hEGF in our study. To explore the possibility of hEGF expression in peanut, hEGF overexpression hairy roots were obtained by infecting leaves with Agrobacterium rhizogenes R1601. Results: The maximum transgenic hairy roots inducing rate was 82%. Protein purification and mass spectrometry assays showed that the protein expressed in peanut hairy roots was identified as hEGF. Furthermore, Methylthiazolyldiphenyl-tetrazolium bromide assay showed that hEGF promoted HL-7702 liver cells proliferation, which indicate that hEGF has biological activity and non-toxic on human cells. Conclusion: Our results demonstrate the capacity of peanut hairy root cultures as a controlled, sustainable, and scalable production system that can be induced to produce valued human proteins, such as hEGF.
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Affiliation(s)
- Qingshou Yao
- State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China.
- College of Pharmaceutical Sciences, Binzhou Medicial University, Yantai 264003, China.
| | - Zipeng Yu
- State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China.
| | - Pu Liu
- State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China.
| | - Hao Zheng
- State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China.
| | - Yang Xu
- Shandong Peanut Research Institute, Shandong Academy of Agricultural Sciences, Qingdao 266100, China.
| | - Sixiang Sai
- College of Pharmaceutical Sciences, Binzhou Medicial University, Yantai 264003, China.
| | - Yuyong Wu
- College of Pharmaceutical Sciences, Binzhou Medicial University, Yantai 264003, China.
| | - Chengchao Zheng
- State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an 271018, China.
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Yu J, Yan Y, Luan X, Qiao C, Liu Y, Zhao D, Xie B, Zheng Q, Wang M, Chen W, Shen C, He Z, Hu X, Huang X, Li H, Shao Q, Chen X, Zheng B, Fang J. Srlp is crucial for the self-renewal and differentiation of germline stem cells via RpL6 signals in Drosophila testes. Cell Death Dis 2019; 10:294. [PMID: 30931935 PMCID: PMC6443671 DOI: 10.1038/s41419-019-1527-z] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/10/2018] [Revised: 03/11/2019] [Accepted: 03/14/2019] [Indexed: 12/22/2022]
Abstract
Self-renewal and differentiation in germline stem cells (GSCs) are tightly regulated by the stem cell niche and via multiple approaches. In our previous study, we screened the novel GSC regulatory gene Srlp in Drosophila testes. However, the underlying mechanistic links between Srlp and the stem cell niche remain largely undetermined. Here, using genetic manipulation of the Drosophila model, we systematically analyze the function and mechanism of Srlp in vivo and in vitro. In Drosophila, Srlp is an essential gene that regulates the self-renewal and differentiation of GSCs in the testis. In the in vitro assay, Srlp is found to control the proliferation ability and cell death in S2 cells, which is consistent with the phenotype observed in Drosophila testis. Furthermore, results of the liquid chromatography-tandem mass spectrometry (LC-MS/MS) reveal that RpL6 binds to Srlp. Srlp also regulates the expression of spliceosome and ribosome subunits and controls spliceosome and ribosome function via RpL6 signals. Collectively, our findings uncover the genetic causes and molecular mechanisms underlying the stem cell niche. This study provides new insights for elucidating the pathogenic mechanism of male sterility and the formation of testicular germ cell tumor.
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Affiliation(s)
- Jun Yu
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Yidan Yan
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Xiaojin Luan
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Chen Qiao
- Department of Clinical Pharmacy, the Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Yuanyuan Liu
- Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Jiangsu, 215002, China
| | - Dan Zhao
- Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Center for Reproduction, The Fourth People's Hospital of Zhenjiang, Zhenjiang Jiangsu, 212013, China
| | - Bing Xie
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Qianwen Zheng
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Min Wang
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Wanyin Chen
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Cong Shen
- Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Jiangsu, 215002, China
| | - Zeyu He
- Department of Clinical Medicine, China Medical University, Shenyang Liaoning, 110001, China
| | - Xing Hu
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China
| | - Xiaoyan Huang
- State Key Laboratory of Reproductive Medicine, Department of Histology and Embryology, Nanjing Medical University, Nanjing Jiangsu, 211166, China
| | - Hong Li
- Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Jiangsu, 215002, China
| | - Qixiang Shao
- Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China.,Department of Immunology and Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang Jiangsu, 212013, China
| | - Xia Chen
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China. .,Reproductive Sciences Institute of Jiangsu University, Zhenjiang Jiangsu, 212001, China.
| | - Bo Zheng
- Center for Reproduction and Genetics, Suzhou Municipal Hospital, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Jiangsu, 215002, China.
| | - Jie Fang
- Department of Gynecology, The Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang Jiangsu, 212001, China.
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Yu J, Luan X, Yan Y, Qiao C, Liu Y, Zhao D, Xie B, Zheng Q, Wang M, Chen W, Shen C, He Z, Hu X, Huang X, Li H, Chen B, Zheng B, Chen X, Fang J. Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila. FASEB J 2019; 33:8125-8137. [PMID: 30921522 DOI: 10.1096/fj.201802536rr] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
The ribonucleoprotein (RNP) spliceosome machinery triggers the precursor RNA splicing process in eukaryotes. Major spliceosome defects are implicated in male infertility; however, the underlying mechanistic links between the spliceosome and the ribosome in Drosophila testes remains largely unresolved. Small ribonucleoprotein particle protein SmD3 (SmD3) is a novel germline stem cell (GSC) regulatory gene identified in our previous screen of Drosophila testes. In the present study, using genetic manipulation in a Drosophila model, we demonstrated that SmD3 is required for the GSC niche and controls the self-renewal and differentiation of GSCs in the testis. Using in vitro assays in Schneider 2 cells, we showed that SmD3 also regulates the homeostasis of proliferation and apoptosis in Drosophila. Furthermore, using liquid chromatography-tandem mass spectrometry methods, SmD3 was identified as binding with ribosomal protein (Rp)L18, which is a key regulator of the large subunit in the ribosome. Moreover, SmD3 was observed to regulate spliceosome and ribosome subunit expression levels and controlled spliceosome and ribosome function via RpL18. Significantly, our findings revealed the genetic causes and molecular mechanisms underlying the stem cell niche and the crosstalk between the spliceosome and the ribosome.-Yu, J., Luan, X., Yan, Y., Qiao, C., Liu, Y., Zhao, D., Xie, B., Zheng, Q., Wang, M., Chen, W., Shen, C., He, Z., Hu, X., Huang, X., Li, H., Chen, B., Zheng, B., Chen, X., Fang, J. Small ribonucleoprotein particle protein SmD3 governs the homeostasis of germline stem cells and the crosstalk between the spliceosome and ribosome signals in Drosophila.
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Affiliation(s)
- Jun Yu
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China.,Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China
| | - Xiaojin Luan
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China.,Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China
| | - Yidan Yan
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China.,Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China
| | - Chen Qiao
- Department of Clinical Pharmacy, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China
| | - Yuanyuan Liu
- Center for Reproduction and Genetics, Suzhou Municipal Hospital-The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
| | - Dan Zhao
- Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China.,Center for Reproduction, The Fourth Affiliated Hospital of Jiangsu University-The Fourth People's Hospital of Zhenjiang, Zhenjiang, China
| | - Bing Xie
- Department of Obstetrics and Gynecology, The Fourth Affiliated Hospital of Jiangsu University-The Fourth People's Hospital of Zhenjiang, Zhenjiang, China
| | - Qianwen Zheng
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China.,Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China
| | - Min Wang
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China
| | - Wanyin Chen
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China
| | - Cong Shen
- Center for Reproduction and Genetics, Suzhou Municipal Hospital-The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
| | - Zeyu He
- Department of Clinical Medicine, China Medical University, Shenyang, China
| | - Xing Hu
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China
| | - Xiaoyan Huang
- Department of Histology and Embryology, State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing, China
| | - Hong Li
- Center for Reproduction and Genetics, Suzhou Municipal Hospital-The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
| | - Binghai Chen
- Department of Urology, The Affiliated Hospital of Jiangsu University
| | - Bo Zheng
- Center for Reproduction and Genetics, Suzhou Municipal Hospital-The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou, China
| | - Xia Chen
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China.,Reproductive Sciences Institute, Jiangsu University, Zhenjiang, China
| | - Jie Fang
- Department of Gynecology, The Affiliated Hospital of Jiangsu University-Jiangsu University, Zhenjiang, China
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Gao J, Li Y, Wan Y, Hu T, Liu L, Yang S, Gong Z, Zeng Q, Wei Y, Yang W, Zeng Z, He X, Huang SH, Cao H. A Novel Postbiotic From Lactobacillus rhamnosus GG With a Beneficial Effect on Intestinal Barrier Function. Front Microbiol 2019; 10:477. [PMID: 30923519 PMCID: PMC6426789 DOI: 10.3389/fmicb.2019.00477] [Citation(s) in RCA: 163] [Impact Index Per Article: 27.2] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Accepted: 02/25/2019] [Indexed: 12/12/2022] Open
Abstract
It has long been known that probiotics can be used to maintain intestinal homeostasis and treat a number of gastrointestinal disorders, but the underlying mechanism has remained obscure. Recently, increasing evidence supports the notion that certain probiotic-derived components, such as bacteriocins, lipoteichoic acids, surface layer protein and secreted protein, have a similar protective role on intestinal barrier function as that of live probiotics. These bioactive components have been named 'postbiotics' in the most recent publications. We previously found that the Lactobacillus rhamnosus GG (LGG) culture supernatant is able to accelerate the maturation of neonatal intestinal defense and prevent neonatal rats from oral Escherichia coli K1 infection. However, the identity of the bioactive constituents has not yet been determined. In this study, using liquid chromatography-tandem mass spectrometry analysis, we identified a novel secreted protein (named HM0539 here) involved in the beneficial effect of LGG culture supernatant. HM0539 was recombinated, purified, and applied for exploring its potential bioactivity in vitro and in vivo. Our results showed that HM0539 exhibits a potent protective effect on the intestinal barrier, as reflected by enhancing intestinal mucin expression and preventing against lipopolysaccharide (LPS)- or tumor necrosis factor α (TNF-α)-induced intestinal barrier injury, including downregulation of intestinal mucin (MUC2), zonula occludens-1 (ZO-1) and disruption of the intestinal integrity. Using a neonatal rat model of E. coli K1 infection via the oral route, we verified that HM0539 is sufficient to promote development of neonatal intestinal defense and prevent against E. coli K1 pathogenesis. Moreover, we further extended the role of HM0539 and found it has potential to prevent dextran sulfate sodium (DSS)-induced colitis as well as LPS/D-galactosamine-induced bacterial translocation and liver injury. In conclusion, we identified a novel LGG postbiotic HM0539 which exerts a protective effect on intestinal barrier function. Our findings indicated that HM0539 has potential to become a useful agent for prevention and treatment of intestinal barrier dysfunction- related diseases.
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Affiliation(s)
- Jie Gao
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Yubin Li
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Yu Wan
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Tongtong Hu
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Liting Liu
- Department of Medical Microbiology and Immunology, Dali University, Dali, China
| | - Shaojie Yang
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Zelong Gong
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Qing Zeng
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Yi Wei
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Weijun Yang
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Zhijie Zeng
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Xiaolong He
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
| | - Sheng-He Huang
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China.,Saban Research Institute, Children's Hospital Los Angeles, University of Southern California, Los Angeles, CA, United States
| | - Hong Cao
- Department of Microbiology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou, China
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Qin X, Pi X, Wang W, Han G, Zhu L, Liu M, Cheng L, Shen JR, Kuang T, Sui SF. Structure of a green algal photosystem I in complex with a large number of light-harvesting complex I subunits. NATURE PLANTS 2019; 5:263-272. [PMID: 30850820 DOI: 10.1038/s41477-019-0379-y] [Citation(s) in RCA: 94] [Impact Index Per Article: 15.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/05/2018] [Accepted: 02/04/2019] [Indexed: 05/05/2023]
Abstract
Photosystem I (PSI) is a highly efficient natural light-energy converter, and has diverse light-harvesting antennas associated with its core in different photosynthetic organisms. In green algae, an extremely large light-harvesting complex I (LHCI) captures and transfers energy to the PSI core. Here, we report the structure of PSI-LHCI from a green alga Bryopsis corticulans at 3.49 Å resolution, obtained by single-particle cryo-electron microscopy, which revealed 13 core subunits including subunits characteristic of both prokaryotes and eukaryotes, and 10 light-harvesting complex a (Lhca) antennas that form a double semi-ring and an additional Lhca dimer, including a novel four-transmembrane-helix Lhca. In total, 244 chlorophylls were identified, some of which were located at key positions for the fast energy transfer. These results provide a firm structural basis for unravelling the mechanisms of light-energy harvesting, transfer and quenching in the green algal PSI-LHCI, and important clues as to how PSI-LHCI has changed during evolution.
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Affiliation(s)
- Xiaochun Qin
- School of Biological Science and Technology, University of Jinan, Jinan, China
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
| | - Xiong Pi
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Wenda Wang
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
| | - Guangye Han
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
| | - Lixia Zhu
- School of Biological Science and Technology, University of Jinan, Jinan, China
| | - Mingmei Liu
- School of Biological Science and Technology, University of Jinan, Jinan, China
| | - Linpeng Cheng
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China
| | - Jian-Ren Shen
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China
- Institute for Interdisciplinary Science, Okayama University, Okayama, Japan
| | - Tingyun Kuang
- Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
| | - Sen-Fang Sui
- State Key Laboratory of Membrane Biology, Beijing Advanced Innovation Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China.
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Zhang YZ, Chen YF, Wu YQ, Si JJ, Zhang CP, Zheng HQ, Hu FL. Discrimination of the entomological origin of honey according to the secretions of the bee (Apis cerana or Apis mellifera). Food Res Int 2019; 116:362-369. [DOI: 10.1016/j.foodres.2018.08.049] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Revised: 08/14/2018] [Accepted: 08/18/2018] [Indexed: 01/07/2023]
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Gu J, Hou D, Li Y, Chao H, Zhang K, Wang H, Xiang J, Raboanatahiry N, Wang B, Li M. Integration of proteomic and genomic approaches to dissect seed germination vigor in Brassica napus seeds differing in oil content. BMC PLANT BIOLOGY 2019; 19:21. [PMID: 30634904 PMCID: PMC6329107 DOI: 10.1186/s12870-018-1624-7] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/20/2018] [Accepted: 12/28/2018] [Indexed: 05/23/2023]
Abstract
BACKGROUND Rapeseed (Brassica napus, B. napus) is an important oil seed crop in the world. Previous studies showed that seed germination vigor might be correlated with seed oil content in B. napus, but the regulation mechanism for seed germination has not yet been explained clearly. Dissecting the regulation mechanism of seed germination and germination vigor is necessary. RESULTS Here, proteomic and genomic approaches were used to analyze the germination process in B. napus seeds with different oil content. The identification of 165 differentially expressed proteins (DEPs) in the germinating seeds of B. napus with high and low oil content was accomplished by two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE). The comparative proteomic results revealed that seeds with high oil content had higher metabolic activity, especially for sulfur amino acid metabolism. Thirty-one unique genes were shown to be significantly changed during germination between the seeds with high and low oil content, and thirteen of these genes were located within the confidence interval of germination-related quantitative trait locus (QTLs), which might play an important role in regulating seed germination vigor. CONCLUSIONS The present results are of importance for the understanding of the regulation mechanism for seed germination vigor in B. napus.
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Affiliation(s)
- Jianwei Gu
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Institute of New Socialist Countryside Development, Hubei Engineering University, Xiaogan, China
| | - Dalin Hou
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University, Huanggang, China
| | - Yonghong Li
- Hybrid Rapeseed Research Center of Shaanxi Province, Shaanxi Rapeseed Branch of National Centre for Oil Crops Genetic Improvement, Yangling, China
| | - Hongbo Chao
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Kai Zhang
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Hao Wang
- Hybrid Rapeseed Research Center of Shaanxi Province, Shaanxi Rapeseed Branch of National Centre for Oil Crops Genetic Improvement, Yangling, China
| | - Jun Xiang
- Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University, Huanggang, China
| | - Nadia Raboanatahiry
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
| | - Baoshan Wang
- College of Life Science, Shandong Normal University, Jinan, China
| | - Maoteng Li
- Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, China
- Hubei Key Laboratory of Economic Forest Germplasm Improvement and Resources Comprehensive Utilization, Hubei Collaborative Innovation Center for the Characteristic Resources Exploitation of Dabie Mountains, Huanggang Normal University, Huanggang, China
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Tang X, Xie X, Wang X, Wang Y, Jiang X, Jiang H. The Combination of piR-823 and Eukaryotic Initiation Factor 3 B (EIF3B) Activates Hepatic Stellate Cells via Upregulating TGF-β1 in Liver Fibrogenesis. Med Sci Monit 2018; 24:9151-9165. [PMID: 30556540 PMCID: PMC6319143 DOI: 10.12659/msm.914222] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
BACKGROUND Piwi-interacting RNA (piRNA) is the largest class of small non-coding RNA, which has also been identified in somatic tissues, and aberrant expression of piRNAs in tumor tissues may be implicated in carcinogenesis. piR-823 is increased in liver cirrhosis and hepatocellular carcinoma (HCC). However, there is no report on the function of piR-823 in hepatic stellate cells (HSCs) activation during hepatic fibrosis. The present study investigated the role of piR-823 in HSC activation. MATERIAL AND METHODS Liver fibrosis was induced in mice by carbon tetrachloride (CCL4) injection and bile duct ligation (BDL). The primary HSCs were isolated from mice and cultured. The expression of piR-823 was measured by real-time PCR. The effect of piR-823 on HSCs was evaluated by either sense sequence or antisense sequence of piR-823 carried by liposome. Proteins binding to piR-823 were assayed by RNA pull-down technique and liquid chromatography-mass spectrometry (LC-MS). RESULTS Our data for the first time show that piR-823 is significantly upregulated in activated HSCs. Overexpression of piR-823 promoted HSC proliferation, α-SMA and COL1a1 production, whereas inhibition of piR-823 suppressed the activity of HSCs. Interestingly, the combination of piR-823 and EIF3B promoted TGF-β1 expression. CONCLUSIONS Our data illustrate a novel mechanism of piR-823 in HSC activities. The combination of piR-823 and EIF3B increased TGF-β1 expression, which activates HSCs in liver fibrosis. piR-823 may be a new target in the treatment of liver fibrosis.
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Affiliation(s)
- Xuechan Tang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
| | - Xiaoli Xie
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
| | - Xin Wang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
| | - Yan Wang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
| | - Xiaoyu Jiang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
| | - Huiqing Jiang
- Department of Gastroenterology, The Second Hospital of Hebei Medical University, Hebei Key Laboratory of Gastroenterology, Hebei Institute of Gastroenterology, Shijiazhuang, Hebei, China (mainland)
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Yang RQ, Chen YL, Chen F, Wang H, Zhang Q, Liu GM, Jin T, Cao MJ. Purification, Characterization, and Crystal Structure of Parvalbumins, the Major Allergens in Mustelus griseus. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2018; 66:8150-8159. [PMID: 29969026 DOI: 10.1021/acs.jafc.8b01889] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/08/2023]
Abstract
Fish play important roles in human nutrition and health, but also trigger allergic reactions in some population. Parvalbumin (PV) represents the major allergen of fish. While IgE cross-reactivity to PV in various bony fish species has been well characterized, little information is available about allergens in cartilaginous fish. In this study, two shark PV isoforms (named as SPV-I and SPV-II) from Mustelus griseus were purified. Their identities were further confirmed by mass spectroscopic analysis. IgE immunoblot analysis showed that sera from fish-allergic patients reacted to both SPV-I and SPV-II, but the majority of sera reacted more intensely to SPV-I than SPV-II. Thermal denaturation monitored by CD spectrum showed that both of the SPV allergens are highly thermostable. SPV-I maintained its IgE-binding capability after heat denaturation, while the IgE-binding capability of SPV-II was reduced. The results of crystal structure showed that SPV-I and SPV-II were similar in their overall tertiary structure, but their amino acid sequences shared lower similarities, indicating that the differences in the IgE-binding capabilities of SPV-I and SPV-II might be due to differential antigen epitopes in these two isoforms.
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Affiliation(s)
- Ru-Qing Yang
- College of Food and Biological Engineering , Jimei University , Xiamen , Fujian 361021 , China
| | - Yu-Lei Chen
- College of Food and Biological Engineering , Jimei University , Xiamen , Fujian 361021 , China
| | - Feng Chen
- Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center , University of Science & Technology of China , Hefei 230007 , China
| | - Heqiao Wang
- Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center , University of Science & Technology of China , Hefei 230007 , China
| | - Qian Zhang
- College of Food and Biological Engineering , Jimei University , Xiamen , Fujian 361021 , China
| | - Guang-Ming Liu
- College of Food and Biological Engineering , Jimei University , Xiamen , Fujian 361021 , China
- Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources , Xiamen , Fujian 361100 , China
| | - Tengchuan Jin
- Hefei National Laboratory for Physical Sciences at Microscale, CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Life Sciences and Medical Center , University of Science & Technology of China , Hefei 230007 , China
| | - Min-Jie Cao
- College of Food and Biological Engineering , Jimei University , Xiamen , Fujian 361021 , China
- Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources , Xiamen , Fujian 361100 , China
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Chen H, Diao X, Wang H, Zhou H. An integrated metabolomic and proteomic study of toxic effects of Benzo[a]pyrene on gills of the pearl oyster Pinctada martensii. ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY 2018; 156:330-336. [PMID: 29573723 DOI: 10.1016/j.ecoenv.2018.03.040] [Citation(s) in RCA: 24] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Revised: 03/11/2018] [Accepted: 03/13/2018] [Indexed: 06/08/2023]
Abstract
Benzo[a]pyrene (BaP) is one of the most important polycyclic aromatic hydrocarbons (PAHs), which are widely present in the marine environment. Because of its teratogenic, mutagenic, and carcinogenic effects on various organisms, the toxicity of BaP is of great concern. In this study, we focused on the toxic effects of BaP (1 µg/L and 10 µg/L) on gills of the pearl oyster Pinctada martensii using combined metabolomic and proteomic approaches. At the metabolome level, the high concentration of BaP mainly caused abnormal energy metabolism, osmotic regulation and immune response marked by significantly altered metabolites in gills. At the proteome level, both concentrations of BaP mainly induced signal transduction, transcription regulation, cell growth, stress response, and energy metabolism. Overall, the research demonstrated that the combination of proteomic and metabolomic approaches could provide a significant way to elucidate toxic effects of BaP on P. martensii.
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Affiliation(s)
- Hao Chen
- State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China; Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China.
| | - Xiaoping Diao
- State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China; Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China.
| | - Haihua Wang
- State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China; Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China.
| | - Hailong Zhou
- State Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Haikou 570228, China; Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China.
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