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ObjectivePhosphatidylinositol 3 kinases (PI3Ks) play an important role in cell directional movement by regulating F-actin. However, the structure and function of PI3Ks are complex. The role of PI3Ks in cell electrotaxis is not fully understood. Therefore, in this study, the model organism Dictyostelium discoideum cells were used as experimental materials to explore the role of PI3K1 and PI3K2 in electrotaxis. MethodsFirstly, PI3K1 coding gene pikA knockout mutant and PI3K2 coding gene pikB knockout mutant were constructed by CRISPR/Cas9 system. Secondly, two mutants were placed in a DC electric field with a strength of 12 V/cm and the electrotaxis were analyzed. ResultsData analysis showed that the direction index of wild-type cells in DC electric field was (0.86±0.03), while the direction index of pikA- and pikB- mutants in DC electric field was (0.95±0.02) and (0.94±0.03), respectively. In addition, the average trajectory speed of wild-type cells in the electric field was (3.34±0.08) μm/min, while the average trajectory speed of pikA- and pikB- mutants were (4.85±0.20) μm/min and (5.48±0.15) μm/min, respectively. The t test showed that there were significant differences in the directedness index and speed between the mutant and wild type. Western blot results showed that both phosphorylated Akt and phosphorylated ERK were significantly increased in pikA- and pikB- mutants. ConclusionPI3K1 and PI3K2 may inhibit the electrotaxis of Dictyostelium discoideum cells by increasing the activity of Akt and ERK.
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OBJECTIVE： To establish a method for measuring the paclobutrazol residue in Ophiopogon japonicus from Sichuan and detect the quality of O. japonicus from Sichuan from different sources. METHODS： Totally 50 batches of samples were collected from different origin places， commercial markets and manufacturers. The sample pretreatment method was QuEChERS method， .ie the sample was extracted by aqueous acetonitrile， salted out by QuEChERS extract package （containing anhydrous magnesium sulfate and anhydrous sodium acetate）， the extract solution was purified by QuEChERS purification package （containing anhydrous magnesium sulfate， N-propyl ethylenediamine， octadecylsilane chemically bonded silica， silica gel， graphitized carbon black） and then added into internal standard triphenyl phosphate. The paclobutrazol residue in O. japonicus from Sichuan was determined by GC-MS/MS. The determination was performed on DB-5MS column. The temperature programming was adopted， and the detector was triple quadrupole MS detector. The initial flow rate of carrier gas was 1.3 mL/min； acquisition mode was MRM. Injection method was splitless injection. RESULTS： The linear range of paclobutrazol was 1.01-505 ng/mL （r＝ 0.999 7）. RSDs of precision， stability （24 h） and repeatability tests were 3.94％， 13.62％， 7.54％ （n＝6）， respectively. Average method recovery was 111.26％ （RSD＝5.43％， n＝9）. The paclobutrazol residue in 50 batches of sample were 0.02-2.72 mg/kg. CONCLUSIONS： Established method is simple， accurate， sensitive and reproducible. It also can be used for the determination of paclobutrazol residue in O. japonicus from Sichuan. The contents of paclobutrazol residue in O. japonicus from Sichuan from different sources are different greatly.
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The theory in the Yellow Emperor’s Internal Classic originates from Chinese ancient philosophy, and to a large extent guided and influenced by natural philosophy, forming the basic theoretic frame of TCM. The article makes analysis on materialism and dialectics.