A novel and highly efficient production system for recombinant adeno-associated virus vector

Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy.     

Precise Reconstruction of Veins and Bile Ducts in Rat Liver Transplantation

Rat orthotopic liver transplantation (ROLT) remains a technically demanding procedure, especially regarding the reconstruction of the suprahepatic vena cava (SHVC). In this study, a new microsuture technique was developed for anastomosis of the SHVC, and a special single-groove cuff and blade-cut stent were introduced. With these modified techniques, we aimed to make a precise anastomosis of the SHVC and to provide optimal cuffs and stents for the reconstruction of the veins and bile ducts. According to different microsuture techniques for the SHVC and different types of cuffs and stents, three ROLT groups were created to compare the operation times and prognoses. Sham operations were performed as controls in the fourth group. The time expenditures with each step were compared among the transplantation groups. Biochemical parameters were tested at the end of a 1-month observation period. The short- and long-term survival rates of the transplantation groups were recorded and compared. Our new microsuture technique was faster than the conventional continuous suture technique for SHVC anastomosis (P < 0.05). The use of a single-groove cuff for reconstruction of the portal vein and the infrahepatic vena cava shortened the anastomotic time (P < 0.05). The use of blade-cut stents resulted in fewer biliary complications and better survival over the short and long terms (P < 0.05). Our new microsuture technique and the single-groove cuffs proved to be a precise method for venous reconstruction which shortened the anhepatic time and the anastomotic time significantly. The blade-cut stents apparently reduced the incidence of biliary complications. In summary, with this precise microsuture technique and delicate cuffs and stents, excellent long-term survival can be achieved easily and stably for ROLT.     

中国大鲵研究进展

中国大鲵是我国特有濒危的两栖物种,是研究生物进化、生物多样性、性别决定分子机制等问题的好材料。近年来,人们对它的研究力度不断加大,著述颇多。本文对大鲵的生态保护、解剖发育、生理生化、遗传进化等方面的近期研究资料进行了整理和回顾,也简要探讨了今后大鲵研究的主要工作,以期为研究者们提供有价值的资料。     

CTSB promotes porcine preadipocytes differentiation by degrading fibronectin and attenuating the Wnt/β-catenin signaling pathway

The process of preadipocytes differentiation plays a vital role in adipose tissue expansion and many factors are involved in this event. Cathepsin B (CTSB), secreted from lysosome, has been reported in regulating a variety of physiological processes. In this study, we demonstrated CTSB promotes lipid accumulation and adipogenic genes expression in porcine primary preadipocytes by degrading fibronectin (Fn), a key component of extracellular matrix. Lithium chloride (LiCl) is an activator of Wnt/β-catenin signaling through stabilizing β-catenin. We found that CTSB can relieve the anti-adipogenic effects of LiCl, indicating that CTSB could impact Wnt/β-catenin signaling pathway. Interestingly, Fn is an important target gene of Wnt/β-catenin. So we considered that CTSB promote preadipocytes differentiation by suppressing these two pathways.     

木本植物内源生长素免疫胶体金定位分析技术的研究

以木本植物杨树(Populus sp.)和核桃(Juglans regia L.)为材料,对内源生长素免疫胶体金定位技术在固定、烤片、免疫染色、显色等关键环节进行了改进优化与验证.结果显示,优化后适合木本植物定位方法的主要技术要点是:在染色中,通过采用尿素-胰蛋白酶联合消化技术和增加牛血清白蛋白处理大大地改善了抗原修复结果,提高了染色的敏感性和特异性.利用优化后的方法对核桃幼胚和杨树嫩茎诱导生根过程中的吲哚乙酸(IAA)进行定位研究,发现杨树试管嫩茎生根过程中,形成层及周缘维管束有很强的IAA信号,核桃子叶生根中,胚中有很强的IAA信号,胚根中有半圆形、胚芽中有>"形强信号区,胚轴信号较弱,胚根信号最强.研究表明,与传统免疫染色方法相比,优化后的方法对木本植物生长素定位具有敏感性高,特异性强,银颗粒明显,背景清晰,耗时少等特点.     

Ageing related thyroid deficiency increases brain-targeted transport of liver-derived ApoE4-laden exosomes leading to cognitive impairment

Alzheimer’s disease (AD) is the prevalent cause of dementia in the ageing world population. Apolipoprotein E4 (ApoE4) allele is the key genetic risk factor for AD, although the mechanisms linking ApoE4 with neurocognitive impairments and aberrant metabolism remains to be fully characterised. We discovered a significant increase in the ApoE4 content of serum exosomes in old healthy subjects and AD patients carrying ApoE4 allele as compared with healthy adults. Elevated exosomal ApoE4 demonstrated significant inverse correlation with serum level of thyroid hormones and cognitive function. We analysed effects of ApoE4-containing peripheral exosomes on neural cells and neurological outputs in aged or thyroidectomised young mice. Ageing-associated hypothyroidism as well as acute thyroidectomy augmented transport of liver-derived ApoE4 reach exosomes into the brain, where ApoE4 activated nucleotide-binding oligomerisation domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome by increasing cholesterol level in neural cells. This, in turn, affected cognition, locomotion and mood. Our study reveals pathological potential of exosomes-mediated relocation of ApoE4 from the periphery to the brain, this process can represent potential therapeutic target.Subject terms: Cognitive neuroscience, Alzheimer''s disease, Cellular neuroscience     

Fluvoxamine increased glutamate release by activating both 5-HT(3) and sigma-1 receptors in prelimbic cortex of chronic restraint stress C57BL/6 mice

Emerging evidence from therapeutic trials in humans and animal models suggests that in the treatment of depression, antidepressants play a role by targeting the glutamatergic system. Fluvoxamine is one of the widely used SSRIs which has been considered to target monoamine neurotransmitter reuptake mechanisms. However, whether fluvoxamine has an effect on the glutamate release is still unclear. The present experiment studied the effect of fluvoxamine on presynaptic glutamate release in prelimbic cortex, both in control C57BL/6 mice and chronic restraint stress C57BL/6 mice, and further investigated the mechanism underlying this effect by using patch clamp, on-line fluorimetry, pharmacological approaches combined with other techniques. The results showed that fluvoxamine increased the glutamate release in the depression model mice but it had no effect on the glutamate release in the control mice. The mechanism underlying these effects in depression model mice was that, fluvoxamine firstly activated presynaptic 5-HT(3) receptors, which transiently increased the Ca(2+) concentration. The increase of Ca(2+) concentration via 5-HT(3) receptors caused the activation of sigma-1 receptors, which were activated by fluvoxamine. The activation of sigma-1 receptors increased the intrasynaptosomal Ca(2+) concentration significantly through the outflow of endoplasmic reticulum calcium and finally activated PKC. These results suggested that fluvoxamine may have a selective effect and different mechanism based on the condition of animal.     

Proteomics of larval hemolymph in Bombyx mori reveals various nutrient-storage and immunity-related proteins

The silkworm, Bombyx mori, is an important economic insect for its production of silk. The larvae of many lepidopteran insects are major agricultural pests and often silkworm is explored as a model organism for other lepidopteran pest species. The hemolymph of caterpillars contains a lot of nutrient and immune components. In this study, we applied liquid chromatography–tandem mass spectrometry to gain a better understanding of the larval hemolymph proteomics in B. mori. We identified 752 proteins in hemolymph collected from day-4 fourth instar and day-7 fifth instar. Nearly half the identified proteins (49 %) were predicted to function as binding proteins and 46 % were predicted to have catalytic activities. Apolipophorins, storage proteins, and 30K proteins constituted the most abundant groups of nutrient-storage proteins. Of them, 30K proteins showed large differences between fourth instar larvae and fifth instar larvae. Besides nutrient-storage proteins, protease inhibitors are also expressed very highly in hemolymph. The analysis also revealed lots of immunity-related proteins, including recognition, signaling, effectors and other proteins, comprising multiple immunity pathways in hemolymph. Our data provide an exhaustive research of nutrient-storage proteins and immunity-related proteins in larval hemolymph, and will pave the way for future physiological and pathological studies of caterpillars.