The protein kinase A/protein kinase G/protein kinase C-family (AGC kinase family) of eukaryotes is involved in regulating numerous biological processes. The 3-phosphoinositide- dependent protein kinase 1 (PDK1), is a conserved serine/threonine kinase in eukaryotes. To understand the roles of PDK1 homologous genes in cell death and immunity in tetraploid Nicotiana tabacum, the previuosly generated transgenic CRISPR/Cas9 lines, in which 5-7 alleles of the 4 homologous PDK1 genes (NtPDK1a/1b/1c/1d homologs) simultaneously knocked out, were used in this study. Our results showed that the hypersensitive response (HR) triggered by transient overexpression of active Pto (Pto^Y207D) or soybean GmMEKK1 was significantly delayed, whereas the resistance to Pseudomonas syrangae pv. tomato DC3000 (Pst DC3000) and tobacco mosaic virus (TMV) was significantly elevated in these partial knockout lines. The elevated resistance to Pst DC3000 and TMV was correlated with the elevated activation of NtMPK6, NtMPK3, and NtMPK4. Taken together, our results indicated that NtPDK1s play a positive role in cell death but a positive role in disease resistance, likely through negative regulation of the MAPK signaling cascade.
Nicotiana/virology* ; Disease Resistance/genetics* ; Plant Diseases/immunology* ; Plants, Genetically Modified/genetics* ; Gene Knockout Techniques ; Plant Proteins/genetics* ; CRISPR-Cas Systems ; Protein Serine-Threonine Kinases/genetics* ; 3-Phosphoinositide-Dependent Protein Kinases/genetics* ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Tobacco Mosaic Virus/pathogenicity*

Autophagy is a highly conserved mechanism for material degradation and recycling in eukaryote cells, and plays important roles in growth, development, stress tolerance and immune responses. ATG10 plays a key role in autophagosome formation. To understand the function of ATG10 in soybean, two homologous GmATG10 genes, namely GmATG10a and GmATG10b, were silenced simultaneously by bean pod mottle virus (BPMV) induced gene silencing. The carbon starvation induced by dark treatment and Western blotting analysis of GmATG8 accumulation level indicated that concurrent silencing GmATG10a/10b resulted in the impairment of autophagy in soybean; disease resistance and kinase assays demonstrated that GmATG10a/10b participated in the immune responses by negatively regulating the activation of GmMPK3/6, indicating that GmATG10a/10b plays a negative regulatory role in immune response in soybean.
Soybeans/genetics* ; Immunity

Warfarin is widely used as an oral anticoagulant in clinic in China. It has narrow therapeutic index, large individual differences on dosage and the variability in warfarin dosage depends on many factors, including gene polymorphism, age, diet habits and so on. For clinicians, how to optimize the individual therapeutic regimen of warfarin is becoming a significant part of their work. In recent years, studies on genetic pharmacogenomics of warfarin are reported frequently. It is found that multiple gene polymorphisms can affect the efficacy of warfarin. We will introduce the gene polymorphism of some warfarin related genes and their effects on warfarin, and the advances of warfarin individual therapeutic regimen will also be discussed here.

The relatively high abundance of geochemical elements such as Nb, Zr, Y and other elements shows serious interferences in the determination of trace silver in geochemical samples by inductively coupled plasma-mass spectrometry ( ICP-MS) . Thus it will lead to large deviation in the determination of geochemical samples without separation and enrichment. The traditional emission spectrum or graphite furnace atomic absorption method is only single-element analysis to the silver and with bad sample representativeness. In this study, load diphenylthiourea ( DPTU) foam selective enrichment was used for the separation of Au and Ag from other interfering elements in geological samples, and thiourea liberation-ICP-MS method was developed for the simultaneous determination of Au and Ag. The samples were first decomposed by 1:1 aqua regia. After addition of 50 mL of water, the samples were adsorbed under oscillation for 30 min at 20℃. The detection limits of the Au and Ag were 0. 02 ng/g and 0. 007μg/g, respectively. The proposed method was successfully applied to the determination of Au and Ag in eight national standard materials.

Objective To study the effects of fluoride on longitudinal growth and pathological changes of cultured rat metatarsal bone rudiments.Methods Twenty-four neonatal SD rats were divided into four groups according to the random number table,then the second,third and fourth metatarsal bone rudiments were surgically isolated.The left metatarsal bone rudiments were cultured in α-MEM without F-as control group and the right metatarsal bone rudiments from the same rat were cultured in α-MEM with 1 × 10-7,1 × 10-6,1 × 10-5 and 1 × 10-4 mol/L F-.The length and width of the mineralized area of metatarsal were measured on day 0,day 1,day 4 and day 7,respectively,and the pathological changes of metatarsal bone rudiments were observed by the routinely paraffin-embedded sections method on day 7.Results On day 7,the length of the mineralized area was significantly lower of right metatarsal bone [(240.5 ± 139.3)μm] than the left metatarsal bone [(394.1 ± 173.9)μm,t =4.37,P ＜ 0.01] in the 1 × 10-4 mol/L F-group,but the width of the mineralized area [(239.9 ± 119.4)μm] was not different significantly compared to the left metatarsal bone [(223.3 ± 99.9)μm,t =0.44,P ＞ 0.05].The relative vertical growth rate of the mineralized area on day 4 was significantly lower of right metatarsal bone [(2.43 ± 1.44)％] than left metatarsal bone [(8.34 ± 1.74)％,t =3.21,P ＜ 0.01] in 1 × 10-4 mol/L F-group,and the difference was also significant on day 7 [(16.16 ± 2.87)％ vs.(26.52 ± 4.46)％,t =3.13,P ＜ 0.01].Toluidine blue staining results showed that the thickness of cartilage cells of proliferation and hypertrophic layers was significantly lower of right metatarsal bone [(111.33 ± 27.29),(125.33 ± 30.08)μm] than left metatarsal bone [(127.33 ± 38.36),(160.50 ± 42.73)μm,t =4.82,5.81,all P ＜ 0.01] in 1 × 104 mol/L F-group.The ratio of proliferative and hypertrophic layers was significantly higher of right metatarsal bone (0.93 ± 0.36) than left metatarsal bone (0.83 ± 0.32,t =4.42,P ＜ 0.01) in 1 × 10-4 mol/L F-group.Conclusions Our findings indicate that excessive fluoride could cause longitudinal bone growth inhibition.Such growth inhibition is mediated by decreased chondrocyte proliferation and differentiation and the disproportion of proliferation and differentiation.

Objective To explore the preparation of specific immune RMA(iRNA) on anti-rabies and further study immunotherapy of rabies virus exposure. Methods Horses were immunized with the rabi-es virus and their livers were isolated from the horse of antiserum, from which total RNA was extracted and purified by sodium lauryisulfonate, phenol, chloroform, ethyiene glycol monomethyl ether, cetyltrimethyam-moniumbromide and alcohol. Results Pure preparation physico-chemical characterization was analyzed, and it's weight was 0.15% of weight of liver. The RNA contained 2.86% DNA and 1.16% protein. The iRNA with a maximum UV absorbance at 258 nm and A_(258/280) about 2.0. The test of RNA was positive, which had a relative molecular mas of 13.7×10~3 by high performance liquid chromatography(HPLC), and its hy-perchromic effect was 50.67%. The vesults of biological activity was showed that the rate of leucocyte adher-ence inhibition(LAI) was 41.73%, The protective rate was 50% and prolonging the life was 31.62%. Conclusion The results obtained with the practical value were identical and provide a basis on medicines of anti-rabies.

Novel method of enantioseparation of ofloxacin by capillary electrophoresis with capactively coupled contactless conductivity detection (C~4D) using binary chiral selectors,hydroxypropyl cyclodextrin and hydroxypropyl methyl cellulose ( HP-β-CD and HPMC) ,was developed. The enantiomers of ofloxacin were baseline separated with an uncoated fused-silica capillary(45 cm×50 m i. d. ,L_(effi) = 40 cm)under the optimum separate conditions, 20 mmol/L HAc +6 mmol/L NaAc + 12 mg/L HPMC +35 mmol/L HP-β-CD,separation voltage +13 kV,electrokinetic injection 11 kV×10 s. The calibration curve of the enantiomers showed good linearity in the range from 0. 8 mg/L to 40 mg/L with LOD =0. 3 mg/L. Effects of several factors on resolutions,such as the composition of the running buffer,the pH value,separation voltage,injection method and the parameters of C~4D detector,were investigated. The efficacy of the HP-β-CD and HPMC were discussed. The proposed method has been successfully applied to monitor the enantiomer of ofloxacin contents in the commercial Ofloacin Tablets and Levofloxacin Hydrochloride Tablets,and was demonstrated simple,rapid and reproducible.