Generation of a recombinant herpes simplex virus which can provide packaging function for recombinant adeno-associated

The generation of a recombinant HSV (rHSV) that can provide packaging function for rAAV production is described. A set of cosmids including cos48, cos28, cos6, cos14 and cos56, which represents the HSV-1 genome was used for generation of this rHSV. Rep and cap genes of AAV-2 were inserted into Xba Ⅰ site of UL2 gene on cos6, generating cos6-rcΔUL2. After being digested with Pac Ⅰ , cos6-rcΔUL2 and the other 4 cosmids were cotrans-fected into BHK-21 cells. The recombinant virus HSV1-rc/ΔUL2 carrying rep and cap genes was generated due to the homologous recombination of the 5 cosmids. The results showed that the existence of rep and cap genes on this rHSV was stable from passage to passage and the rHSV could support the packaging of rAAV either in cells transiently transfected with AAV vector or in stable cell line harboring AAV vector. Further modification of this rHSV and optimization of conditions involved in rAAV preparation may lead to a large-scale production of rAAV in the near future.     

A new type of triploid crucian crap?red crucian carp (♀)×allotetraploid (♂)

A new type of triploid hybrids (TC) was produced by crossing red crucian carp (♀) with allotetraploid hybrids (♂). This type of triploids with no barbel was spindle-shaped and gray in color. Compared with their parents, some data of the morphological traits in the triploid hybrids were intermediate to those of their parents, and some were beyond their parents with heterozygous traits. Under the same culture conditions, the growth rate of the new type triploids was faster than their maternal red crucian carp (RC). The gonadal development of triploids was obviously slower than that of diploids, and the germ cells in them were degenerated. Compared with another type of triploids produced by Japanese crucian carp (♀) with allotetraploid hybrids (♂), the new type triploids not only kept faster growth rate and sterility, but also had no barbel that was similar to the common carp. The absence of the barbel in the new type of triploids has the important significance in both the inheritance and fish breeding.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp (Carassius caassius red var.) and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b) . All of the four Sox9 genes contained an intron in the HMG-box. with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of "GT-AG" . A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

S100A8 mediates the activation of P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinogenesis

S100 calcium binding protein A8 (S100A8),a possible novel member of NF-kappa B signal pathway in laryngeal squamous cell carcinoma (LSCC),interacts with human leukocyte antigen B (HLA-B) which carries an NF-kappa B binding site within the enhancer A. The objective of this study was to explore the molecular mechanism of S100A8 in laryngeal carcinogenesis. RT-PCR,Western blotting and immuno-histochemistry staining were applied to evaluate the expression levels of IKKα,P65,REL-B,S100A8,APAF-1 and BCL-2 genes. The signal transduction passway in which S100A8 might participate was explored by RNA interference. Flow cytometry,TUNEL assay and cell invasion in vitro were used to detect the biological behavior of Hep2 cells induced by S100A8 gene. Our results showed that high expression of S100A8 was related to tumorigenesis in LSCC and negatively correlated with the degree of differentiation,indicating that S100A8 gene could inhibit apoptosis and promote metastasis in LSCC. Additionally,the suppression of S100A8 by RNA interference down-regulated BCL-2 but not APAF-1,P65 and IKKα,while,the suppression of P65 could significantly down-regulate the expression of S100A8 gene. In conclusion,S100A8 plays an important role in P65/HLA-B/S100A8/BCL-2/Caspase-9 (-3) pathway in laryngeal carcinoma.     

Transmembrane structure and function of dctPQM encoding C4－dicarboxylate transport proteins from nitrogen－fixing P. stutzeri A1501

C4-dicarboxylate transport proteins of diazotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ,and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene encoding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii.Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively.The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ2L The resultant plasmid was named pSZY6.By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501,randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was constructed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20,10 and 5mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A150L This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.     

Analysis of intron sequence variability of the conservative HMG-box of Sox9 genes in allotetraploids and their original parents

The Sox genes of allotetraploids and their original maternal red crucian carp （Carassius caassius red var.） and original paternal common carp (Cyprinus carpio L.) were detected by PCR with the designed primers based on the conserved HMG-box sequence in different species. Sequencing of Sox genes indicated that two Sox9 genes (Atsox9a and Atsox9b) existed in allotetraploids, while only one Sox9 gene existed in red crucian carp (Rcsox9a) and common carp (Ccsox9b). All of the four Sox9 genes contained an intron in the HMG-box, with the sizes of 413 bp, 703 bp, 401 bp and 714 bp, respectively. Moreover, the introns obeyed the rule of “GT-AG”. A high similarity was observed between introns of Atsox9a and Rcsox9a (94.4%), Atsox9b and Ccsox9b (97.8%). Interestingly, the deduced amino acid sequences of their corresponding exons all shared 100% identity. Thus, introns of the HMG-domain of Sox9s in allotetraploids and their original parents have not only the length polymorphism but also intron variability. Our results provide significant molecular evidence for the origin and evolution of allotetraploids.     

Production of pig iron from red mud waste fines using thermal plasma technology

Red mud, an insoluble residue produced during alkali leaching of bauxite, is considered as a low-grade iron ore containing 30% to 50% iron. The present paper deals with the use of thermal plasma technology for producing pig iron from red mud waste fines. The smelting reduction of red mud was carried out in a 35 kW DC extended arc thermal plasma reactor. Red mud was properly mixed with fluxes and graphite (fixed carbon, 99%) as a reductant as per stoichiometric requirement. The effect of various process parameters like a reductant, fluxes and smelting time on iron recovery was studied and optimized. An optimum condition for the maximum recovery of iron was obtained. A new thermal plasma process applicable to direct iron making from red mud waste fines that would achieve significant utilization of red mud was proposed.     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

不同单色光对紫色红曲霉生长、色素和桔霉素合成的影响

以紫色红曲霉M9为研究对象,研究不同单色光对其生长、色素和桔霉素合成的影响。采用观察法和高效液相色谱法对紫色红曲霉M9在持续红光、黄光、绿光、蓝光照射下的菌落形态及6种红曲色素产量进行研究。采用高效液相色谱法和RT-qPCR法对不同红光光照时间和光照强度下红曲色素和桔霉素产量以及相关基因表达量进行测定。结果表明,红光是最显著的促进紫色红曲霉M9生长和色素产生的光源。高产红曲色素、低产桔霉素的最佳光照时间和强度分别为30min/d和300lx,初步推测红曲色素合成相关基因mppA/B/D/F、mppR1/R2、MpPKS5、MpFasA2/B2可能参与两种橙色素的生物合成,mppC、mppE可能参与两种红色素和两种黄色素的生物合成;桔霉素合成相关基因ctnA/D/E/F/G/H/I、orf1/3/4/5、pksCT可能参与桔霉素的合成代谢,而ctnR1可能参与桔霉素的分解代谢。     

“红色资源”概念的界定

准确界定红色资源的概念,是构建红色资源学科体系的内在要求,也是促进红色资源开发利用的客观需要。纵观学术界现有对红色资源概念界定的理论成果,主要存在对红色资源的内涵主体认识不统一,对红色资源的时间外延有争议,对红色资源的核心词把握不科学以及没有突出转化在红色资源概念界定中应有的地位等问题。通过分析,可以对红色资源的概念作这样的界定:红色资源是指中国人民在中国共产党领导下,在新民主主义革命到改革开放前创造和形成的,可以为我们今天开发利用,且必须经过转化才能够彰显出其当代价值的革命精神及其载体的总和。     

改性赤泥负载氧化钴催化剂活化过一硫酸盐降解罗丹明B

赤泥是制铝工业排放的强碱性固体废弃物,利用改性赤泥制备类芬顿催化剂可以实现赤泥资源化利用。以酸化改性赤泥为载体,采用浸渍法制备了负载氧化钴催化剂。以罗丹明B为目标物,研究了酸化改性赤泥负载氧化钴催化剂活化过一硫酸盐（peroxymonosulfate,PMS）处理罗丹明B的性能,考察了催化剂用量、PMS浓度、罗丹明B初始浓度以及反应温度的影响。结果表明,在催化剂用量为0.05 g/L、PMS浓度为0.1 mmol/L、溶液初始pH为4.8、反应温度为65 ℃、罗丹明B质量浓度为10 mg/L、反应时间为50 min的条件下,罗丹明B的去除率达到95.9%。淬灭实验结果表明,反应体系中同时存在SO₄^-· 、·OH和单线态氧¹O₂,其中¹O₂的氧化反应起主导作用。4次循环实验后罗丹明B的去除率仍能维持在80.0%以上。     

3个观赏海棠半同胞家系子代叶色动态分析与初选

【目的】探讨观赏海棠自由授粉子代苗期叶色变异规律,为半同胞家系育种及海棠新品种选育提供参考。【方法】以3个绿叶观赏海棠品种(‘冬金'、‘冬红'和‘红珠宝')为母本的自由授粉半同胞家系子代为试材,采用Minolta色差仪对120株半同胞家系子代不同叶位叶片色彩进行动态测定,进行叶色动态变化研究及优选。【结果】①基于5—8月子代上叶位L^*、C^*、h°色彩参数进行CIELCH色空间动态分布图的构建,发现 ‘冬金' 家系子代叶色参数位点最为分散,叶色最为丰富,子代中出现母本不具有的亮绿色和红色叶片。②基于5月子代上位叶色彩参数(L^*、C^*、h°),构建叶色聚类分析图,发现在欧式距离19水平上可将子代划分为两大色系类群和 5 个子色系类群,即绿色系(包括褐绿色、深绿色和亮绿色)和红色系(包括紫红色和棕红色),其中绿色系占比92.5%。③基于聚类分析结果及色彩参数分别构建绿色系和红色系子代评价体系,绿色系中从高亮度、高饱和度及色彩稳定性角度优选出 ‘冬金' 家系的‘A03'、‘A09', ‘冬红' 家系的‘D21'和 ‘红珠宝' 家系的‘C10'、‘C26'、‘C29', 红色系从低色调角和色彩持续性角度优选出‘冬金' 家系的‘A37',优株叶色观赏性优于资源圃品种。【结论】通过自由授粉,观赏海棠半同胞子代群体遗传基础大大增加,叶色多样性得以提高; 以参数色空间作为优选评价方法,更为直观可视化,可用作观赏海棠实践生产及新品种选育等。     

基于LabVIEW的海洋赤潮生物荧光实时采集系统

赤潮是近海常见的重要灾害之一,为了建立对赤潮生物的有效监测方法，本文提出了一种基于LabVIEW语言的海洋赤潮生物荧光实时采集系统．该系统通过光电倍增管将赤潮生物受激光激发出的荧光信号获取并转换为电流信号，通过信号调理电路及基于LabVIEW编程的数据采集卡将电压信号读入计算机处理并分析．实验结果表明,该系统可以实时有效地捕获海水样品中赤潮生物的荧光信号。为检测和计算海洋赤潮生物及其浓度提供了一种有效的辅助检测途径，有助于预测海洋赤潮爆发．     

A point mutation in the A gamma-globin gene promoter in Greek hereditary persistence of fetal haemoglobin

Hereditary persistance of fetal haemoglobin (HPFH) is a benign condition characterized by the production in adulthood of more than 1% fetal haemoglobin (HbF, alpha 2 gamma 2) in the absence of erythropoietic stress. Several genetic types have been discerned based on the level of HbF produced, the relative contributions of the duplicated fetal (G gamma and A gamma) globin genes, and the presence or absence of deletions involving the beta and delta genes in cis to the mutation. Greek HPFH is a non-deletion variety in which heterozygotes produce 10-20% HbF, predominantly due to overproduction of the A gamma chain. We have cloned a 40-kilobase (kb) region of the beta-globin cluster from a Greek HPFH allele and report here that a point mutation (G----A) occurs 117 base pairs (bp) 5' to the cap site of the A gamma-globin gene, just upstream of the distal CCAAT sequence. The corresponding region of the G gamma-globin gene is normal. We discuss the implications of this finding for the developmental regulation of globin gene expression.     

微波诱变选育红色素高产菌株初探

以红色素产生菌W0为出发菌株,通过微波诱变、微波和紫外复合诱变,得到3株产红色素较高的生产菌W1,W2,W3,结果表明,复合诱变比单一因子诱变的效果好,复合诱变筛选到的W3产生红色素的色价由1.073提高到1.748,提高了63%.遗传稳定性实验表明,3株突变株的产红色素的性能较稳定.     

用活性炭吸附和TiO2光催化降解法处理酸性红G染色废水

用活性炭吸附、二氧化钛光催化降解处理酸性红G染色废水．用正交试验法研究了活性炭用量、吸附时间、二氧化钛用量、紫外灯照射时间对酸性红G去除率的影响．结果表明：若使用活性炭吸附,在20mL浓度为5．26×10^-5mol／L的废水中加0．8g活性炭,搅拌30min,废水中酸性红G去除率达97．1％．若用二氧化钛光催化降解,则在20mL相同浓度的废水中加二氧化钛0．05g,在300W紫外灯下照20min,废水中酸性红G去除率为99．2％．     

Parallel colour-opponent pathways to primary visual cortex

The trichromatic primate retina parses the colour content of a visual scene into 'red/green' and 'blue/yellow' representations. Cortical circuits must combine the information encoded in these colour-opponent signals to reconstruct the full range of perceived colours. Red/green and blue/yellow inputs are relayed by the lateral geniculate nucleus (LGN) of thalamus to primary visual cortex (V1), so understanding how cortical circuits transform these signals requires understanding how LGN inputs to V1 are organized. Here we report direct recordings from LGN afferent axons in muscimol-inactivated V1. We found that blue/yellow afferents terminated exclusively in superficial cortical layers 3B and 4A, whereas red/green afferents were encountered only in deeper cortex, in lower layer 4C. We also describe a distinct cortical target for 'blue-OFF' cells, whose afferents terminated in layer 4A and seemed patchy in organization. The more common 'blue-ON' afferents were found in 4A as well as lower layer 2/3. Chromatic information is thus conveyed to V1 by parallel, anatomically segregated colour-opponent systems, to be combined at a later stage of the colour circuit.