Critical role of interleukin-17/interleukin-17 receptor axis in mediating Con A-induced hepatitis

Concanavalin A (Con A)-induced hepatitis is thought to be a T-cell-mediated disease with active destruction of liver cells. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells. However, whether IL-17/IL-17 receptor (IL-17/IL-17R)-mediated responses are involved in T-cell-mediated Con A-induced liver injury remains unclear. In this study, we found that IL-17 expression was highly elevated in liver tissues during Con A-induced hepatitis. The increased levels of IL-17 were paralleled with the severity of liver injury reflected by Alanine aminotransaminase and histological assay as well as the secretion of tumor necrosis factor (TNF)-α and IL-6. Blockage of IL-17 significantly ameliorated Con A-induced hepatitis, while overexpression of IL-17 systemically resulted in massive hepatocyte necrosis in mice. Furthermore, overexpression of an IL-17R immunoglobulin G1 fusion protein significantly attenuated liver inflammation after acute Con A treatment. High expression of IL-17R on Kupffer cells was also observed along with the production of cytokines including TNF-α and IL-6. Inhibition of Kupffer cells by gadolinium chloride completely prevented Con A-induced liver injury and cytokine release. Finally, IL-17-expressing CD4(+) T and natural killer T cells were greatly increased in Con A-injected mice compared with that in controls. Overall, our results indicate that IL-17R signaling is critically involved in the pathogenesis in Con A-induced hepatitis, and blockade of IL-17/IL-17R signaling pathway may represent a novel therapeutic intervention in human autoimmune-related hepatitis.     

MicroRNA-409-3p inhibits migration and invasion of bladder cancer cells via targeting c-Met

There is increasing evidence suggesting that dysregulation of certain microRNAs (miRNAs) may contribute to tumor progression and metastasis. Previous studies have shown that miR-409-3p is dysregulated in some malignancies, but its role in bladder cancer is still unknown. Here, we find that miR-409-3p is down-regulated in human bladder cancer tissues and cell lines. Enforced expression of miR-409-3p in bladder cancer cells significantly reduced their migration and invasion without affecting cell viability. Bioinformatics analysis identified the pro-metastatic gene c-Met as a potential miR-409-3p target. Further studies indicated that miR-409-3p suppressed the expression of c-Met by binding to its 3′-untranslated region. Silencing of c-Met by small interfering RNAs phenocopied the effects of miR-409-3p overexpression, whereas restoration of c-Met in bladder cancer cells bladder cancer cells overexpressing miR-409-3p, partially reversed the suppressive effects of miR-409-3p. We further showed that MMP2 and MMP9 may be downstream effector proteins of miR-409-3p. These findings indicate that miR-409-3p could be a potential tumor suppressor in bladder cancer.     

嗜碱单胞菌的新型羧基转移酶α亚基基因Aa-accA及其抗盐碱性能

[目的]探讨解淀粉嗜碱单胞菌(Alkalimonas amylolytica)N10来源的羧基转移酶α亚基(Acetyl-coenzyme A carboxylase subunit alpha,AccA)基因Aa-accA对细菌及植物细胞耐盐碱性的作用.[方法]通过PCR方法从嗜碱菌N10基因组中扩增基因Aa-accA,并在大肠杆菌(Escherichia coli)K12中表达,通过测定工程菌及对照菌在不同盐浓度[0%,2%,4%,6%(W/V) NaCl]及不同碱性pH(8.0,8.5,9.0,9.5)的LB中生长12 h后的OD600值,以及二者在分别含6%(W/V) NaCl及pH 9的LB中的生长曲线,评价Aa-accA对大肠杆菌耐盐碱性的影响.同时以pPZP111为载体,构建了植物细胞重组表达载体,通过农杆菌介导方法将该基因转入烟草BY-2悬浮细胞表达,利用FDA染色方法测定经盐碱溶液处理后残存的活细胞数量评价该基因对植物细胞耐盐碱性的影响.[结果]PCR扩增得到基因Aa-accA,其ORF含957 bp,编码318个氨基酸的多肽,BLAST比对显示该基因为羧基转移酶α亚基(AccA)家族中的成员,其氨基酸序列与E.coli的AccA具有76%同源性;含有Aa-accA的E.coli K12相较于对照组在不同NaCl浓度及不同碱性pH的LB中表现出了明显的生长优势,特别是在6%(W/V) NaCl及pH 9的LB中培养12 h后,终OD600分别是对照菌的2.6倍和3.5倍;缺失体实验结果显示基因缺失的突变体E.coli K12△accA在6%(W/V) NaCl及pH 9的LB中不能正常生长,而含有Aa-accA基因的重组质粒使得E.coli K12△accA在同样条件下OD600值达到0.5和0.2;转入此基因的烟草BY-2细胞,经盐碱溶液处理后,其存活细胞比例高于野生型.[结论]本研究首次发现了Aa-accA基因与盐碱性的相关性,可提高大肠杆菌及烟草BY-2细胞的耐盐碱能力.     

Flowering-Related RING Protein 1 (FRRP1) Regulates Flowering Time and Yield Potential by Affecting Histone H2B Monoubiquitination in Rice (Oryza Sativa)

Flowering time is a critical trait for crops cultivated under various temperature/photoperiod conditions around the world. To understand better the flowering time of rice, we used the vector pTCK303 to produce several lines of RNAi knockdown transgenic rice and investigated their flowering times and other agronomic traits. Among them, the heading date of FRRP1-RNAi knockdown transgenic rice was 23–26 days earlier than that of wild-type plants. FRRP1 is a novel rice gene that encodes a C3HC4-type Really Interesting Novel Gene (RING) finger domain protein. In addition to the early flowering time, FRRP1-RNAi knockdown transgenic rice caused changes on an array of agronomic traits, including plant height, panicle length and grain length. We analyzed the expression of some key genes associated with the flowering time and other agronomic traits in the FRRP1-RNAi knockdown lines and compared with that in wild-type lines. The expression of Hd3a increased significantly, which was the key factor in the early flowering time. Further experiments showed that the level of histone H2B monoubiquitination (H2Bub1) was noticeably reduced in the FRRP1-RNAi knockdown transgenic rice lines compared with wild-type plants and MBP-FRRP1-F1 was capable of self-ubiquitination. The results indicate that Flowering Related RING Protein 1 (FRRP1) is involved in histone H2B monoubiquitination and suggest that FRRP1 functions as an E3 ligase in vivo and in vitro. In conclusion, FRRP1 probably regulates flowering time and yield potential in rice by affecting histone H2B monoubiquitination, which leads to changes in gene expression in multiple processes.     

Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic <Emphasis Type="Italic">Halomonas</Emphasis> sp. Y2

Na⁺/H⁺ antiporters play important roles in ion and pH homeostasis. In this study, two NhaD homologues that effectively catalyze Na⁺/H⁺ antiporter were identified from Halomonas sp. Y2, a halotolerant and alkaliphilic strain isolated from sodium enriched black liquor. They exhibited high sequence identity of 72 % and similar binding affinities for Na⁺ and Li⁺ translocation, while having different pH profiles. Ha-NhaD1 was active at pH 6.0 and most active at pH 8.0–8.5, whereas Ha-NhaD2 lacked activity at pH 6.0 but exhibited maximum activity at pH 9.5 or higher. Based on multiple alignments, 11 partially conserved residues were selected and corresponding mutants were generated for Ha-NhaD1. As expected, replacement of most of the hydrophobic residues abolished the cation exchange activities. Three serine residues at positions 200, 282 and 353 in Ha-NhaD1 were replaceable by alanines with partial retention of activity. The S353A mutant exhibited significantly reduced binding affinity for Na⁺ and Li⁺, while S282 mutant exhibited an alkaline shift of about 1.5 pH units, as compared to the wild type Ha-NhaD1. Serine at position 282 was predicted to be located in transmembrane segment VIII and was found to be important in regulating pH sensitivity in concert with flanking residues.     

The Protein Phosphorylation Landscape of Mouse Spermatids during Spermiogenesis

The characteristic tadpole shape of sperm is formed from round spermatids via spermiogenesis, a process which results in dramatic morphological changes in the final stage of spermatogenesis in the testis. Protein phosphorylation, as one of the most important post‐translational modifications, can regulate spermiogenesis; however, the phosphorylation events taking place during this process have not been systematically analyzed. In order to better understand the role of phosphorylation in spermiogenesis, large‐scale phosphoproteome profiling is performed using IMAC and TiO₂ enrichment. In total, 13 835 phosphorylation sites, in 4196 phosphoproteins, are identified in purified mouse spermatids undergoing spermiogenesis in two biological replicates. Overall, 735 testis‐specific proteins are identified to be phosphorylated, and are expressed at high levels during spermiogenesis. Gene ontology analysis shows enrichment of the identified phosphoproteins in terms of histone modification, cilium organization, centrosome and the adherens junction. Further characterization of the kinase‐substrate phosphorylation network demonstrates enrichment of phosphorylation substrates related to the regulation of spermiogenesis. This global protein phosphorylation landscape of spermiogenesis shows wide phosphoregulation across a diverse range of processes during spermiogenesis and can help to further characterize the process of sperm generation. All MS data are available via ProteomeXchange with the identifier PXD011890.     

Identification and immunological evaluation of novel TLR2 agonists through structure optimization of Pam3CSK4

Toll-like receptor 2 (TLR2) is a bridge between innate immunity and adaptive immunity. TLR2 agonists have been exploited as potential vaccine adjuvants and antitumor agents. However, no TLR2 agonists have been approved by FDA up to now. To discover drug-like TLR2 selective agonists, a novel series of Pam₃CSK₄ derivatives were designed based on the crystal structure of hTLR2-hTLR1-Pam₃CSK₄ complex, synthesized and evaluated for their immune-stimulatory activities. Among them, 35c was identified as a murine-specific TLR2 agonist, while 35f was a human-specific TLR2 agonist. Besides, 35d (human and murine TLR2 agonist) showed TLR2 agonistic activity comparable to Pam₃CSK₄, which included: elevated IL-6 expression level (EC₅₀ = 83.08 ± 5.94 nM), up-regulated TNF-α and IL-6 mRNA expression and promoted maturation of DCs through activating the NF-κB signaling pathway. TLRs antibodies test showed that 35a and 35d were TLR2/1 agonists, while 35f was a TLR2/6 agonist.     

Cotton CSLD3 restores cell elongation and cell wall integrity mainly by enhancing primary cellulose production in the Arabidopsis cesa6 mutant

Key message

Overexpression of cotton cellulose synthase like D3 (GhCSLD3) gene partially rescued growth defect of atcesa6 mutant with restored cell elongation and cell wall integrity mainly by enhancing primary cellulose production.

Abstract

Among cellulose synthase like (CSL) family proteins, CSLDs share the highest sequence similarity to cellulose synthase (CESA) proteins. Although CSLD proteins have been implicated to participate in the synthesis of carbohydrate-based polymers (cellulose, pectins and hemicelluloses), and therefore plant cell wall formation, the exact biochemical function of CSLD proteins remains controversial and the function of the remaining CSLD genes in other species have not been determined. In this study, we attempted to illustrate the function of CSLD proteins by overexpressing Arabidopsis AtCSLD2, -3, -5 and cotton GhCSLD3 genes in the atcesa6 mutant, which has a background that is defective for primary cell wall cellulose synthesis in Arabidopsis. We found that GhCSLD3 overexpression partially rescued the growth defect of the atcesa6 mutant during early vegetative growth. Despite the atceas6 mutant having significantly reduced cellulose contents, the defected cell walls and lower dry mass, GhCSLD3 overexpression largely restored cell wall integrity (CWI) and improved the biomass yield. Our result suggests that overexpression of the GhCSLD protein enhances primary cell wall synthesis and compensates for the loss of CESAs, which is required for cellulose production, therefore rescuing defects in cell elongation and CWI.