[Investigation of Lentiviral Vectors Based Integrin ß8 RNAi System in Neonatal Rats' Brain].

OBJECTIVE: To construct lentiviral vectors expressing pSicoR-ß8 shRNA and evaluate its efficiency of RNA interference in neonatal rats' brain. METHODS: Plasmid vectors pSicoR-ß8 shRNA and pSicoR-control,as well as lentiviral packaging system pDM2G,g/p RRE and pRSV Rev were amplified respectively and plasmid DNA was identified by restriction enzyme digestion. Lentiviral packaging system and expressing vector pSicoR-ß8 shRNA/pSicoR-control were co-transfected into packaging cell line 293T. Lentiviral particles expressing ß8-shRNA or control sequence packaged and secreted by 293T were collected,concentrated by PEG-it,and viral titers were assayed by 50% tissue culture infective dose (TCID50). RNAi for integrin ß8 in neonatal rats' brain was performed by intraventricular injection of lentivirus expressing ß8-shRNA and rats received lentivirus expressing ß8-shRNA were served as control. Green fluorescent protein (GFP) expression after intraventricular injection of GFP-Lentivirus was observed under fluorescence microscope,ß8 mRNA and ß8 protein expression were detected by RT-PCR and Western blot respectively,all of which were performed to evaluate the RNAi efficiency and to choose the optimal time for intervention. RESULTS: Restrictive endonuclease digestion and agarose gel electrophoresis showed plasmids as same as the expected size. Lentiviral titers for LV-control after concentration was 1.0×108 PFU/mL,and for LV-ß8 shRNA 5.0×108 PFU/mL.One day after intraventricular injection of lentiviral vectors containing GFP sequence,lenticivirus genome was integrated into host cells and emitted green fluorescence. A relatively strong green fluorescence could be observed in brain slides 2 d,3 d and 5 d after intraventricular injection. Western blot and RT-PCR demonstrated a maximum inhibition happened 3 d after intraventricular injection of LV-ß8 shRNA,the inhibitory rate for ß8 mRNA and ß8 protein were 56% and 51%,respectively. CONCLUSION: Lentiviral vectors expressing ß8-shRNA are successfully constructed and lentiviral mediated ß8-RNAi is successfully applied for in vivo use.

[Comparison of soil heavy metals determined by AAS/AFS and portable X-ray fluorescence analysis].

Total concentrations of Cu, Pb, As, Cr, Ni and Zn were determined for 53 soil samples using portable X-ray fluorescence (PXRF) system in in-situ and ex-situ (Lab.) conditions. PXRF metal concentrations were statistically compared with analytical results from traditional AAS/AFS analysis. The ability of PXRF instrument to produce comparable analytical results to the reference method was assessed by linear regression. To investgate the effects of soil moisture on PXRF, the in-situ moisture content of all soil samples was quantified and the metal concentrations of selected samples with known moisture contents were measured too. The results showed that the detection limits of PXRF for Cu, Pb, As, Cr, Ni and Zn were 10.6, 8.1, 5.7, 22.5, 21.6 and 10.4 mg kg(-1) respectively. A good degree of linearity was found for Pb, Cr, Ni and Zn in in-situ condition. While in ex-situ condition, quantitative level data were achieved across the entire range of samples tested for Cu, Pb, Cr, Ni and Zn. X-ray fluorescence spectrometry was shown to be an effective tool for quantification and rapid assessment of heavy metals in soils. Soil moisture content did affected the performance of PXRF, the mean percent difference for soil samples in-situ with moisture content less than 15% and higher than 25% was -17% and -31% respectively. In ex-situ condition, as the soil moisture content increased from air dried level to 30%, the mean percent difference decreased from 10% to -24%. The dilution effect of moisture in soils may cause discrepancies with conventional analytical results and induce worse data quality, and it should be controlled within 0-25% in in-situ condition.

4-(2,4-Dichloro-phen-yl)-5,5-dimethyl-2-(3-silatranyl-propyl-mino)-1,3,2-dioxa-phospho-rinane 2-oxide.

In the title compound, C(20)H(31)Cl(2)N(2)O(6)PSi, the dioxaphospho-rinane ring adopts a cis conformation. The silatrane fragment forms a cage-like structure in which there exists an intra-molecular Si-N donor-acceptor bond. In the crystal, centrosymmetrically related mol-ecules are linked by pairs of N-H⋯O hydrogen bonds into inversion dimers, generating rings with graph-set motif R(2) (2)(8). The dimers are further connected into ribbons parallel to the a axis by inter-molecular C-H⋯O hydrogen bonds.

[Effect of ginsenoside Rg1 on angiogenesis after neonatal hypoxia ischemia brain damage in rats].

OBJECTIVE: To investigate effects of ginsenoside Rg1 on angiogenesis in neonatal rats with hypoxia ischemia brain damage (HIBD), and explore the possible mechanism. METHODS: Fifty-four of 10-day-old SD rats were randomly divided into sham-operation group (n = 6), hypoxia-ischemia brain damage group (n = 24) and ginsenoside Rg1 treatment group (n = 24). SD rats in HIBD group and Rg1 group were treated by separation and ligation of right common carotid artery (CCA) and subsequently exposed to hypoxia for 2.5 hours, and those in sham group were treated by only separation of right CCA, without ligation or exposure to hypoxia. Intraperitoneal injection of 0.1 mL normal saline (NS) containing 40 mg/kg Rg1 was performed immediately after operation in Rg1 group, and such process was repeated every 24 h for 3 days. Intraperitoneal injection of 0.1 mL pure NS was performed in both HIBD group and sham group, in the same way as that of in Rg1 group. General state of SD rats after operation was monitored, 4, 8, 24 and 72 hours after HIBD, animals were executed and the right side of brain tissue was separated for further process. Protein expression of both hypoxia inducible factor 1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) were detected by both Western blot and immunohistochemistry. Immunohistochemistry for von willebrand factor (vwf) was used to labeling micro vessels. RESULTS: All rats survived to the end of the study and neurological dysfunction was observed in both HIBD group and Rgl group, but not in sham group. Expression of HIF-1alpha protein in HIBD group was increased at 4, 8, 24 and 72 h, compared to that in sham group (P < 0.05). Expression of HIF-1alpha protein in Rg1 group was increased compared to that in HIBD group at the same time points (P < 0.05). Expression of VEGF protein in HIBD group was increased at 4, 8, and 24 h, compared to that in sham group (P < 0.05). Expression of VEGF protein in Rg1 group was increased at 24 and 72 h compared to that in HIBD group at the same time point (P < 0.05). Number of vwf-positive cells at 24 and 72 h in HIBD group was increased compared to that in sham group (P < 0.05), and number of vwf-positive cells at 72 h in Rg1 group was increased compared to that in HIBD group at the same time point (P < 0.05). CONCLUSION: Rg1 could facilitate angiogenesis after HIBD in Neonatal rats by strengthening and stabilizing HIF-1alpha/VEGF signaling pathway.

Network pharmacology and in vitro study of Fengreqing oral liquid in the intervention of wind-heat pattern.

OBJECTIVE: To investigate the underlying mechanism of the effect of Fengreqing oral liquid (, FOL) on wind-heat pattern (WHP). METHODS: In this study, we predicted the potential targets of FOL via the approach of network pharmacology and verified it by in vitro inflammation model. In the network pharmacology part, two strategies, namely the direct target search and the indirect one, were used to collect the target sets of FOL in WHP treatment. The enrichment analysis was carried out by David database and ClueGo plug-in in Cytoscape. Furthermore, the potential targets were mapped in the candidate pathways. In the verification experiment section, in vitro model of lipopolysaccharide (LPS) induced RAW 264.7 was used to confirm the predictive results in the network pharmacology part. RESULTS: Through the two screening strategies, a total of 141 non-repetitive intervention targets of FOL on WHP were obtained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed that the intervention effect was mainly focused on the anti-inflammatory effect, and the Toll-like receptor signaling pathway was one of the most critical regulatory pathways. Further mapping analysis showed that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling transfer might be the key part of regulating the concentration of inflammation mediators of FOL in the Toll-like receptor signaling pathway. In vitro experiment showed that FOL significantly reduced the levels of NO, IL-1, IL-6, and TNF-α produced by RAW264.7 induced by LPS. Further immunofluorescence found that this effect is related to the regulation of PI3K-AKT pathway activity by FOL. CONCLUSION: FOL can intervene in WHP by regulating the content of inflammatory mediators via the PI3K-AKT pathway.

[Comparing the ammonia volatilization characteristic of two typical paddy soil with total wheat straw returning in Taihu Lake region].

An experiment using monolith lysimeter was conducted to compare the characteristic of N loss by ammonia (NH3) volatilization between the gleyed paddy soil (G soil) and hydromorphic paddy soil (H soil) the Changshu National Agro-ecological Experimental Station of the Chinese Academy of Sciences(31 degrees 33' N, 123 degrees 38' E). Three treatments were designed for each soil type, i. e. control (no urea and straw applied), nitrogen solely and nitrogen plus wheat straw. Ammonia volatilization, flood water NH4(+) -N concentration, pH and top soil Eh were measured during the rice-growing season. Results showed that the NH3 volatilization flux and cumulative N losses by NH3 volatilization from G soil were significantly higher than those from H soil, the average cumulative N losses being about 41.8 kg x hm(-2) and 11.2 kg x hm(-2), or 15.2% and 3.8% of the fertilizer N, respectively. The average N loss by NH3 volatilization during the tillering stage was the highest among the three fertilization stages, accounting for 29.4% and 8.3% of the fertilizer N for G soil and H soil, respectively. Wheat straw returning significantly increased paddy filed NH3 volatilization losses. Comparing with the sole application of fertilizer-N, the cumulative N loss by NH3 volatilization of fertilizer-N in combination with wheat straw was increased by 19.8% and 20.6% for G soil and H soil, respectively. In addition, ammonia volatilization fluxes showed a positive relationship with the flood water NH4(+) -N concentration and pH for both soils, but the relationship with top soil Eh still needs further study.

[Effects of long-term manure and crop residues incorporation on yield and phosphorus saturation in a paddy soil].

An 8-year field experiment was conducted in the Taihu Lake region of eastern China to investigate the effects of incorporation of straw and manure on the yield and phosphorus (P) accumulation in the paddy soil, and to evaluate the potential risk of P loss from soil to environment. The experiment had four fertilization treatments, i. e., chemical fertilizers alone (NPK), chemical fertilizers plus rice/wheat straw (NPK + S), chemical fertilizers plus 7.5 t x (hm2 x a)(-1) wet pig manure (NPK + M7.5), and chemical fertilizers plus 15.0 t x (hm2 x a)(-1) wet pig manure (NPK + M15). Among the four treatments, no significant differences were observed in the yield of rice or wheat. Long-term application of chemical fertilizers plus pig manure significantly increased the soil total P, the degree of P saturation (DPS), and the concentration of extractable P forms, including Olsen-P, Mehlich 3 extractable P, CaCl2 extractable P, and water extractable P, which became a potential source of eutrophication in Taihu Lake. In contrast to chemical fertilizers plus pig manure, there were no significant differences in the concentrations of extractable P forms between the NPK + S and NPK treatments. We concludes that chemical fertilizers [P 45 kg x (hm2 x a)(-1)] plus rice/wheat straw should be recommended in the paddy soil in the Taihu Lake region under the rice-wheat rotation system.