Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways.

RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

Effects of betamethasone on neuropathic pain in a rat spare nerve injury model.

The aim of the present study was to examine the effect of glucocorticoids on neuropathic pain using a rat spare nerve injury (SNI) model. Eighty rats were treated divided into the following groups: (i) a sham-operated group; (ii) a group subjected to SNI (S); (iii) a group subjected to SNI and administered 4 µg betamethasone intrathecally (D1); and (iv) a group subjected to SNI and administered 1 mg betamethasone at the site of nerve injury (D2). The mechanical withdrawal threshold (MWT) and thermal withdrawal duration (TWD) were measured 1 day before and the 1, 3, 7 and 14 days after SNI. Glial fibrillary acidic protein, glucocorticoid receptor (GR), tumour necrosis factor (TNF)-α and interleukin (IL)-1ß levels in spinal cord tissue were quantified 1, 3, 7 and 14 days after SNI. The MWT was significantly higher in the D2 compared with S group 3-14 days after surgery and compared with the D1 group 7 and 14 days after surgery (P < 0.05). The TWD was significantly lower in the D2 group compared with the S and D2 groups 3-14 days after surgery (P < 0.05). Glial fibrillary acidic protein expression was significantly lower in the D1 and D2 groups compared with the S group 3-14 days after surgery (P < 0.05). Glucocorticoid receptor expression was significantly higher in the D1 group compared with the S and D2 groups after surgery (P < 0.05). Levels of TNF-α and IL-1ß were significantly lower in the D1 and D2 groups compared with the S group at all time points after surgery (P < 0.05). Betamethasone suppressed astrocyte activation and increases in TNF-α and IL-1ß levels in a rat model of neuropathic pain. Local injection of betamethasone resulted in smaller increases in spinal GR expression and more pronounced improvement in pain behaviour compared with intrathecal injection.

[Monitoring the change in CO concentration in combustion with tunable diode laser absorption spectroscopy].

In the present paper, the technology of tunable diode laser absorption spectroscopy (TDLAS) in conjunction with the open path multi-pass Herriot cell and the new-style detection method of auto-balanced detection combined with wavelength modulation technology were used, and the concentration of CO produced in combustion of alcohol blowtorch was measured. It was found in the measured result that the change in CO concentration in the flame of alcohol blowtorch presented a stated periodicity in the process of combustion and the average concentration of CO was calculated to be 49.4 (10(-6) ratio by volume). The experiment is showed that with the conjunction of auto-balanced detection and the second harmonics detection method, adopting the open path multi-pass Herrriot cell to detect the concentration of CO in the combustion of alcohol blowtorch is accurate and contents the detection requirement. It was proved that the system made for measuring the concentration of CO in the flame of alcohol blowtorch in combustion establishes foundation well for developing on-line combustion monitoring based on TDLAS.

[Second-harmonic detection with tunable diode laser absorption spectroscopy of CO and CO2 at 1.58 microm].

Tunable diode laser absorption spectroscopy has been applied in the fields of atmospheric chemistry and monitoring pollutant gases as a new method of measuring trace gases. The technique of remote sensing of CO and CO2 at 760 mm Hg pressure with tunable diode laser absorption spectroscopy in the near-infrared region is introduced. And the relationship between the second-harmonic spectrum of CO2 in Lorentzian line shape and the modulation amplitude is also presented.

[Selection of recombinant fowlpox virus coexpressing HIV-1 gag-gp120 and IL-6].

OBJECTIVE: To construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6. METHODS: The recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed. RESULTS: There was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity. CONCLUSION: The recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.