The mRNA–miRNA–lncRNA Regulatory Network and Factors Associated with Prognosis Prediction of Hepatocellular Carcinoma

The aim of this study was to identify novel prognostic mRNA and microRNA (miRNA) biomarkers for hepatocellular carcinoma (HCC) using methods in systems biology. Differentially expressed mRNAs, miRNAs, and long non-coding RNAs (lncRNAs) were compared between HCC tumor tissues and normal liver tissues in The Cancer Genome Atlas (TCGA) database. Subsequently, a prognosis-associated mRNA co-expression network, an mRNA–miRNA regulatory network, and an mRNA–miRNA–lncRNA regulatory network were constructed to identify prognostic biomarkers for HCC through Cox survival analysis. Seven prognosis-associated mRNA co-expression modules were obtained by analyzing these differentially expressed mRNAs. An expression module including 120 mRNAs was significantly correlated with HCC patient survival. Combined with patient survival data, several mRNAs and miRNAs, including CHST4, SLC22A8, STC2, hsa-miR-326, and hsa-miR-21 were identified from the network to predict HCC patient prognosis. Clinical significance was investigated using tissue microarray analysis of samples from 258 patients with HCC. Functional annotation of hsa-miR-326 and hsa-miR-21-5p indicated specific associations with several cancer-related pathways. The present study provides a bioinformatics method for biomarker screening, leading to the identification of an integrated mRNA–miRNA–lncRNA regulatory network and their co-expression patterns in relation to predicting HCC patient survival.     

左旋千金藤啶碱D1激动—D2阻滞作用引起伏核双相放电活动的电生理研究

为确定左旋千金藤啶碱（ＳＰＤ）对中脑边缘ＤＡ神经系统的作用特性,本研究采用细胞外记录的电生理学方法,观察微电泳和尾静脉给药对６－ＯＨＤＡ损毁及未损毁大鼠的伏核（ＮＡｃ）单位放电的影响。结果显示：ＳＰＤ累积给药（０．０２－２ｍｇ／ｋｇ,ｉｖ）可诱发ＮＡｃ神经元双相放电特征,即小剂量抑制、大剂量兴奋。预先给予Ｄ２受体拮抗剂ｓｐｅｐｅｒｏｎｅ,ＳＰＤ则仅产生兴奋效应,并被Ｄ１拮抗剂ＳＣＨ－２３３９０所翻     

On the origin of the Norwegian lemming

The Pleistocene glacial cycles resulted in significant changes in species distributions, and it has been discussed whether this caused increased rates of population divergence and speciation. One species that is likely to have evolved during the Pleistocene is the Norwegian lemming (Lemmus lemmus). However, the origin of this species, both in terms of when and from what ancestral taxon it evolved, has been difficult to ascertain. Here, we use ancient DNA recovered from lemming remains from a series of Late Pleistocene and Holocene sites to explore the species' evolutionary history. The results revealed considerable genetic differentiation between glacial and contemporary samples. Moreover, the analyses provided strong support for a divergence time prior to the Last Glacial Maximum (LGM), therefore likely ruling out a postglacial colonization of Scandinavia. Consequently, it appears that the Norwegian lemming evolved from a small population that survived the LGM in an ice‐free Scandinavian refugium.     

Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker

A chromosome-plasmid balanced lethal gene delivery system for Lactobacillus acidophilus based on the thyA gene was developed. The selected L. acidophilus DOM La strain carries a mutated thyA gene and has an obligate requirement for thymidine. This strain can be used as a host for the constructed shuttle vector pFXL03, lacking antibiotic-resistant markers but having the wild-type thyA gene from L. casei which complements the thyA chromosomal mutation. The vector also contains the replicon region from plasmid pUC19 and that of the Lactococcus plasmid pWV01, which allows the transfer between Escherichia coli, L. casei and L. acidophilus. Eight unique restriction sites (i.e., PstI, HindIII, SphI, SalI, AccI, XbaI, KpnI and SacI) are available for cloning. After 40-time transfers in modified MRS medium, no plasmid loss was observed. The vector pFXL03 is potentially useful as a food-grade vaccine delivery system for L. acidophilus.     

利用短短小芽孢杆菌启动子和信号肽编码序列构建穿梭分泌表达载体

以短短小芽孢杆菌B15的总DNA为模板,利用PCR技术克隆到其细胞壁蛋白基因串联启动子和信号肽编码序列,测序分析后提交GenBank,登录号为AY956423。重新设计引物扩增该片段并在PCR产物两侧引入BamHⅠ和PstⅠ酶切位点,将PCR产物双酶切后克隆至穿梭载体pP43NMK的相应位点构建分泌表达载体pP15MK,插入片段置于该载体中mpd基因的上游,并使信号肽编码序列与去除了自身信号肽编码序列的mpd基因阅读框恰好融合。将pP15MK导入枯草杆菌构建表达菌株1A751(pP15MK),在短短小芽孢杆菌启动子和信号肽元件的带动下,mpd基因能够在表达菌株的对数生长期和稳定期持续性高效分泌表达,表达产物结合在细胞膜上;发酵液在48h酶活达到最高值7.79U/mL,是出发菌株邻单胞菌M6表达量的8.1倍。     

Cloning and biological activity of an anti-tumor peptide of Tumstatin

To obtain an anti-tumor peptide of Tumstatin and detect its biological activity, the nucleotide sequence encoding 185–203 amino acids (19peptide) of Tumstatin was synthesized and inserted into the fusion protein vector pTYB2. After identification by sequencing and restriction endonucleases, the recombined vector was transformed into BL-21 (DE3) E. coli competent cells. Transformed E. coli BL-21 (DE3) were induced by isopropyl-β-thiogalactopyranoside (IPTG), and then expressed. By 1,4-dithiothreitol (DTT) reduction, the soluble 19peptide was obtained from a chitin affinity chromatograph. The biological activity of 19peptide was determined by 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenytetrazolium bromide (MTT) assay, cell growth curve, the effect of the ascitic fluid transfevent H22 hepatoma on mice and via histopathological slices. The purified 19peptide directly inhibited proliferation and migration of murine B16 melanoma cells, SMMC-7721hepatoma carcinoma cells and human umbilical vein endothelial cells (HUVEC). The tumor inhibition rate of mice ascitic fluid transfevent H22 hepatoma was 48.46%. Histopathological slices showed that it could promote tumor tissue necrosis and decrease the density of blood vessels. With higher anti-tumor activity, 19peptide has the potential to become a novel, potent anti-tumor agent. Translated from Chinese Journal of Biochemistry and Molecular Biology, 2005, 21(3): 322–328 [译自: 中国生物化学与分子生物学学报]     

TNFα-initiated oxidative/nitrative stress mediates cardiomyocyte apoptosis in traumatic animals

Whole body non-penetrating trauma causes myocardial infarction in humans and mechanical trauma (MT) results in cardiac dysfunction in animals. Our recent study demonstrated that incubation of cardiomyocytes with plasma isolated from MT animals causes significant cardiomyocyte apoptosis that can be blocked by neutralization of TNFα. The present study attempted to obtain direct in vivo evidence to support that overproduction of TNFα plays a causative role in trauma-induced cardiomyocyte apoptosis. Non-lethal MT caused significant TNFα overproduction (2.4-fold at 1.5 h after MT) and increased cardiomyocyte apoptosis (starting 3 h and peaking 12 h after MT). Pharmacological inhibition of TNFα with etanercept or TNFα gene deletion reduced post-trauma myocyte apoptosis (P < 0.01). Expression of iNOS and NADPH oxidase, overproduction of NO and , and excessive protein nitration in the MT heart were all significantly reduced in etanercept-treated or TNFα^−/− mice, suggesting that oxidative/nitrative stress may contribute to TNFα-initiated myocyte apoptosis in MT hearts. Additional experiments demonstrated that inhibiting iNOS (1400W) or NADPH oxidase (apocynin), or scavenging peroxynitrite (FP15) significantly reduced myocyte apoptosis in MT animals (P < 0.01). Collectively, these data demonstrated that non-lethal mechanical trauma caused significant TNFα production that in turn stimulated myocardial apoptosis via oxidative/nitrative stress.     

A highly sensitive biosensor with (Con A/HRP)n multilayer films based on layer-by-layer technique for the detection of reduced thiols

The bilayer of Con A/HRP through the biospecific affinity of concanavalin A (Con A) and glycoprotein horseradish peroxidase (HRP) was prepared on the surface of an Au electrode modified by the precursor film consisted of poly(allylamine hydrochloride) poly(sodium-p-styrene-sulfonate). Atomic force microscopy and electrochemical impedance spectroscopy were adopted to monitor the uniform layer-by-layer assembly of the Con A/HRP bilayers. The amperometric measurement was based on the inhibition of reduced thiols and performed in the presence of the electron mediator hydroquinone in 0.2 M phosphate buffer of pH 6.5 at an applied potential of −0.15 V versus Ag/AgCl. Under the optimal conditions, the biosensor presented a linear response for cysteine from 0.1 to 23.5 μM, with a detection limit of 0.02 μM. The biosensor demonstrated high stability and repeatability. A series of reduced thiols were detected by this inhibition biosensor and oxidized thiols showed no effect on the current response of the biosensor.