Recovery of DNA from agarose gel by trap method

Recovery of DNA from agarose gel electrophoresis is a basic operation during molecular cloning. Circular or linear DNA fragments which vary from 1.5 to 6.5 kb and correspond to 1 kb marker can be recovered from 0.8 to 1.0% agarose gel smoothly with a simple and rapid trap method. The recovery efficiency could be more than 70% and the quality of the recovered DNA is proved to be good enough for future research. Here, we provided another good method to recover DNA from agarose gel besides using commercial kits.Key words: Trap method, recovery of DNA, agarose gel electrophoresis

Mesh reinforced membrane and its wrinkling characteristics

Membrane structures have received a wide range of attention in applications of large-scale spacecrafts, such as inflated wing, light-than-air (LTA) airship and membrane antenna reflector, and so on. These spacecrafts need to be designed as high loading-efficiency components or structures, especially with high shape precision [1]. Therefore, some special processing and design should be done on the membranes so as to satisfy with special requirements, such as free wrinkle, ultralightweight, high shape precision, and high load-carrying ability, etc.. Several applications may give us good ideas to deal with abovementioned problems in membrane structures. One example is easy to be remembered, that is, the Super-Pressure Balloons (SPB) [2-4] covered by some ropes on membrane surface to make the balloon stable and strong. The similar considerations are also applied to some gossamer spacecraft components or structures, taking the Lunar habitat [5] as an example

ZIKV infection activates the IRE1-XBP1 and ATF6 pathways of unfolded protein response in neural cells.

BACKGROUND: Many viruses depend on the extensive membranous network of the endoplasmic reticulum (ER) for their translation, replication, and packaging. Certain membrane modifications of the ER can be a trigger for ER stress, as well as the accumulation of viral protein in the ER by viral infection. Then, unfolded protein response (UPR) is activated to alleviate the stress. Zika virus (ZIKV) is a mosquito-borne flavivirus and its infection causes microcephaly in newborns and serious neurological complications in adults. Here, we investigated ER stress and the regulating model of UPR in ZIKV-infected neural cells in vitro and in vivo. METHODS: Mice deficient in type I and II IFN receptors were infected with ZIKV via intraperitoneal injection and the nervous tissues of the mice were assayed at 5 days post-infection. The expression of phospho-IRE1, XBP1, and ATF6 which were the key markers of ER stress were analyzed by immunohistochemistry assay in vivo. Additionally, the nuclear localization of XBP1s and ATF6n were analyzed by immunohistofluorescence. Furthermore, two representative neural cells, neuroblastoma cell line (SK-N-SH) and astrocytoma cell line (CCF-STTG1), were selected to verify the ER stress in vitro. The expression of BIP, phospho-elF2α, phospho-IRE1, and ATF6 were analyzed through western blot and the nuclear localization of XBP1s was performed by confocal immunofluorescence microscopy. RT-qPCR was also used to quantify the mRNA level of the UPR downstream genes in vitro and in vivo. RESULTS: ZIKV infection significantly upregulated the expression of ER stress markers in vitro and in vivo. Phospho-IRE1 and XBP1 expression significantly increased in the cerebellum and mesocephalon, while ATF6 expression significantly increased in the mesocephalon. ATF6n and XBP1s were translocated into the cell nucleus. The levels of BIP, ATF6, phospho-elf2α, and spliced xbp1 also significantly increased in vitro. Furthermore, the downstream genes of UPR were detected to investigate the regulating model of the UPR during ZIKV infection in vitro and in vivo. The transcriptional levels of atf4, gadd34, chop, and edem-1 in vivo and that of gadd34 and chop in vitro significantly increased. CONCLUSION: Findings in this study demonstrated that ZIKV infection activates ER stress in neural cells. The results offer clues to further study the mechanism of neuropathogenesis caused by ZIKV infection

Zika virus promotes CCN1 expression via the CaMKIIα-CREB pathway in astrocytes.

Zika virus (ZIKV) infection in the human central nervous system (CNS) causes Guillain-Barre syndrome, cerebellum deformity, and other diseases. Astrocytes are immune response cells in the CNS and an important component of the blood-brain barrier. Consequently, any damage to astrocytes facilitates the spread of ZIKV in the CNS. Connective tissue growth factor/Nephroblastoma overexpressed gene family 1 (CCN1), an important inflammatory factor secreted by astrocytes, is reported to regulate innate immunity and viral infection. However, the mechanism by which astrocyte viral infection affects CCN1 expression remains undefined. In this study, we demonstrate that ZIKV infection up-regulates CCN1 expression in astrocytes, thus promoting intracellular viral replication. Other studies revealed that the cAMP response element (CRE) in the CCN1 promoter is activated by the ZIKV NS3 protein. The cAMP-responsive element-binding protein (CREB), a transacting factor of the CRE, is also activated by NS3 or ZIKV. Furthermore,a specific inhibitor of CREB, i.e. SGC-CBP30, reduced ZIKV-induced CCN1 up-regulation and ZIKV replication. Moreover, co-immunoprecipitation, overexpression, and knockdown studies confirmed that the interaction between NS3 and the regulatory domain of CaMKIIα could activate the CREB pathway, thus resulting in the up-regulation of CCN1 expression and enhancement of virus replication. In conclusion, the findings of our investigations on the NS3-CaMKIIα-CREB-CCN1 pathway provide a foundation for understanding the infection mechanism of ZIKV in the CNS

Establishment of a high-efficiency plant regeneration and transformation system for the elite maize inbred lines from three heterotic groups

Maize (Zea mays L.) is one of the most important crops in the world and its agronomic traits could be improved by genetic transformation with desirable genes. A successful transformation must depend ona high-efficiency in vitro plant regeneration and genetic transformation system. In our studies, six media compositions were used to induce callus from the immature zygotic embryos of seven maize inbred lines and LM6 was proved the best callus induction media, with high callus induction percentage (CIP) and callus quality. Furthermore, four phytohormones were analyzed on their effects on callus induction, the results indicated that 2,4-dichlorophenoxyacetic acid (2,4-D) played an important role in callusinitiation, but, 3.3 mg/l Dicamba could provide higher embryogenic callus induction percentage (EIP) than 2 mg/l 2,4-D; both 6-benzyl aminopurine (6-BA) and kinetin (KT) could decrease CIP, EIP and callus quality at the concentration level of 0.2 mg/l. The experimental result also revealed that media LM6-CI was preferable to LM6 in callus induction. The selected LM6-CI media, together with other two media (LM6-EI and LM6-PR), were used to identify 18 elite maize inbred lines from three heterotic groups ontheir tissue culture characteristics, as a result, the eight materials, including 3189/4380, 4380/Sanzong5, 8103, Xianzao17, 18-599(red), 501, 178 and Ji53 belonging to group Reid or Compound Germplasm,presented higher CIP, EIP and plant regeneration percentage (PRP) than others; the four materials, including Huangzao4, Huangye4, Jing24 and Ji853 from Tangsipingtou group, were not easy to be differentiated into plants, in spite of high CIP. Maize inbred line 18-599(red) as a representative was further used to establish genetic transformation system, its embryogenic calli, initiated from immature zygotic embryos, were transformed with the plasmid p35SBarNos harboring Bar gene by microprojectile bombardment, after selection and differentiation culture, partial bombarded calli were regenerated into green plantlets and further fertile plants. The results of molecular identification for fertile regeneratedplants showed that Bar gene had been integrated into maize genome and the transformation frequency was high up to 66.7%. All these results were beneficial for the studies on in vitro plant regeneration and genetic transformation in plant