Discovery and Analysis of MicroRNAs in Leymus chinensis under Saline-Alkali and Drought Stress Using High-Throughput Sequencing

Leymus chinensis (Trin.) Tzvel. is a perennial rhizome grass of the Poaceae (also called Gramineae) family, which adapts well to drought, saline and alkaline conditions. However, little is known about the stress tolerance of L. chinensis at the molecular level. microRNAs (miRNAs) are known to play critical roles in nutrient homeostasis, developmental processes, pathogen responses, and abiotic stress in plants. In this study, we used Solexa sequencing technology to generate high-quality small RNA data from three L. chinensis groups: a control group, a saline-alkaline stress group (100 mM NaCl and 200 mM NaHCO₃), and a drought stress group (20% polyethylene glycol 2000). From these data we identified 132 known miRNAs and 16 novel miRNAs candidates. For these miRNAs we also identified target genes that encode a broad range of proteins that may be correlated with abiotic stress regulation. This is the first study to demonstrate differentially expressed miRNAs in L. chinensis under saline-alkali and drought stress. These findings may help explain the saline-alkaline and drought stress responses in L. chinensis.     

The Functions of Crucial Cysteine Residues in the Arsenite Methylation Catalyzed by Recombinant Human Arsenic (III) Methyltransferase

Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs³⁺). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs³⁺ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs³⁺ was methylated to dimethylarsinous acid (DMA³⁺). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs³⁺ methylation catalyzed by hAS3MT.     

Lactoferrin Deficiency Promotes Colitis-Associated Colorectal Dysplasia in Mice

Nonresolving inflammatory processes affect all stages of carcinogenesis. Lactoferrin, a member of the transferrin family, is involved in the innate immune response and anti-inflammatory, anti-microbial, and anti-tumor activities. We previously found that lactoferrin is significantly down-regulated in specimens of nasopharyngeal carcinoma (NPC) and negatively associated with tumor progression, metastasis, and prognosis of patients with NPC. Additionally, lactoferrin expression levels are decreased in colorectal cancer as compared with normal tissue. Lactoferrin levels are also increased in the various phases of inflammation and dysplasia in an azoxymethane–dextran sulfate sodium (AOM-DSS) model of colitis-associated colon cancer (CAC). We thus hypothesized that the anti-inflammatory function of lactoferrin may contribute to its anti-tumor activity. Here we generated a new Lactoferrin knockout mouse model in which the mice are fertile, develop normally, and display no gross morphological abnormalities. We then challenged these mice with chemically induced intestinal inflammation to investigate the role of lactoferrin in inflammation and cancer development. Lactoferrin knockout mice demonstrated a great susceptibility to inflammation-induced colorectal dysplasia, and this characteristic may be related to inhibition of NF-κB and AKT/mTOR signaling as well as regulation of cell apoptosis and proliferation. Our results suggest that the protective roles of lactoferrin in colorectal mucosal immunity and inflammation-related malignant transformation, along with a deficiency in certain components of the innate immune system, may lead to serious consequences under conditions of inflammatory insult.     

Role of Moesin in Renal Fibrosis

Background

Renal fibrosis is the final common pathway of chronic kidney disease (CKD). Moesin is a member of Ezrin/Radixin/Moesin (ERM) protein family but its role in renal fibrosis is not clear.

Method

Human proximal tubular cells (HK-2) were stimulated with or without TGF-β1. Moesin and downstream target genes were examined by real-time PCR and western blot. Phosphorylation of moesin and related signaling pathway was investigated as well. Rat model of unilateral ureteral obstruction (UUO) was established and renal moesin was examined by immunohistochemistry. Moesin in HK-2 cells were knocked down by siRNA and change of downstream genes in transfected HK-2 cells was studied. All animal experiments were reviewed and approved by the Ethics Committee for animal care of Ruijin Hospital.

Result

HK-2 cells stimulated with TGF-β1 showed up-regulated level of α-SMA and down-regulated level of E-Cadherin as well as elevated mRNA and protein level of moesin. In rat model of UUO, renal moesin expression increased in accordance with severity of tubulointerestital fibrosis in the kidneys with ureteral ligation while the contralateral kidneys were normal. Further study showed that TGF-β1 could induce phosphorylation of moesin which depended on Erk signaling pathway and Erk inhibitor PD98059 could block moesin phosphorylation. Effects of TGF-β1 on moesin phosphorylation was prior to its activation to total moesin. RNA silencing studies showed that knocking down of moesin could attenuate decrease of E-Cadherin induced by TGF-β1.

Conclusion

We find that moesin might be involved in renal fibrosis and its effects could be related to interacting with E-Cadherin.     

Survivin小分子抑制剂的研究进展

Survivin是凋亡抑制蛋白家族的一员,在抑制细胞凋亡、调控细胞周期、参与血管形成等方面发挥重要的生物学功能。Survivin在多种肿瘤组织中过量表达,与肿瘤不良预后和耐药性密切相关。Survivin作为一种潜在的肿瘤治疗靶点,其小分子抑制剂用于肿瘤治疗的研究为人们所关注。概述了Survivin的结构、功能及其在肿瘤组织中的特异性表达,综述了目前靶向Survivin的小分子抑制剂的研究进展。     

选择性环氧合酶-2抑制剂联合奥曲肽诱导人胃癌细胞的凋亡

目的探讨选择性环氧合酶-2抑制剂NS-398与奥曲肽联合应用对人胃癌细胞株BGC-823生长、凋亡的影响。方法体外培养BGC-823细胞,分别用NS-398(100μmol/L)与奥曲肽(1μmol/L)单独及联合处理不同时间后,倒置显微镜观察细胞形态学变化;观察生长曲线的变化;流式细胞仪检测细胞凋亡率;实时定量(Real-time)PCR检测COX-2mRNA的表达;Western blot法检测Caspase-3蛋白表达。结果倒置显微镜下,对照组BGC-823细胞生长良好,药物处理后,细胞变小、变圆,悬浮,联合组细胞形态学改变显著强于单纯用药组;药物作用后,细胞生长受抑制,出现负增长,联合组作用明显强于单纯用药组;流式细胞仪检测表明联合用药组诱导BGC-823细胞的凋亡率明显高于单一用药组和对照组(P0.01);各处理组均使BGC-823细胞COX-2mRNA表达下调(P0.05);药物处理后细胞Caspase-3蛋白表达明显增加。结论 NS-398、奥曲肽联合可协同抑制BGC-823细胞生长、增殖,其机制可能与下调COX-2mRNA表达、诱导肿瘤细胞凋亡相关。     

气管导管生物膜培养对病原学检出率的影响

目的探讨机械通气危重儿气管导管细菌生物膜对提高病原菌检出率的作用。方法选择2013年入住新生儿科需要机械通气的新生儿,根据机械通气的时间将研究对象分为3组:≤72 h组、73~168 h组及>168 h组,以漩涡振荡器振荡气管导管末端,振荡液培养,与同时送检的深部痰培养结果进行统计学比较。结果在机械通气时间≤72 h组、73~168 h组中,漩涡振荡器振荡液与深部痰培养的阳性率高度一致,Kappa值分别为0.799、0.710,P值分别为0.000及0.000;在>168 h组中,二者中度一致,Kappa值为0.651,P值为0.001。两种方法分离的菌株以产ESBL酶肺炎克雷伯杆菌最多见,产ESBL酶大肠杆菌次之,两种方法间菌株的构成及耐药性差异无统计学意义。结论以漩涡振荡器法获取气管导管细菌生物膜不能提高机械通气患儿的病原学检测阳性率,对临床用药无明显指导意义。     

大仔买麻藤在两种栽培环境下叶片的营养成分比较

为探讨栽培环境对大仔买麻藤(Gnetum montanum Markgr.f.megalocarpa Markgr.)叶片营养成分的影响,将采自云南省景东县的大仔买麻藤分别在北京和昆明的温室内培养,对其1年生幼苗叶片的营养成分进行研究。结果表明,不同栽培环境对大仔买麻藤叶片的大部分营养成分无显著影响;叶片总淀粉、总糖、黄酮和生物碱含量无显著差异;但在二糖、莰菲醇、部分氨基酸和某些元素含量上有显著差异;并且大部分的元素含量受土壤的影响显著。因此,不同栽培环境对大仔买麻藤叶片营养成分的影响不大。