Objective To establish the determination method of syringin and syringaresinol in Ciwujia Injection with RP-HPLC. Methods C18 ODS was used as a stationary phase. The mobile phase consists of water and acetonitrile. The gradient condition was 0→20 min, A:90%→50%. The flow rate was 1.0 mL/min. The detective wavelength was set at 220 nm. Results The linear range of syringin was 0.03～0.16 ?g (r =0.999 7, n=5) and the linear range of Syringaresinol was 0.02～0.13 ?g (r =0.999 3, n=5). The average recovery of syringin was 99.34% and RSD=0.58%. The average recovery of Syringaresinol was 99.22% and RSD=0.71%. Conclusions The method was simple, convenient and accurate, and can be used for content determination of syringin and syringaresinol in Giwujia injection.

BACKGROUND:Human and rat ovarian granulosa cels in dominant folicles have the phenomenon of expressing stem cel characteristics. OBJECTIVE:To investigate the expression of stem cel-related factors in rat ovarian granulosa cels. METHODS: After the paraffin sections of rat ovarian tissue, immunohistochemical method was used to detect CD34, CD133, ABCG2/Bcrp1, Pou5f1/Oct-4 expressions. Granulosa cels culturedin vitro were harvested by folicular puncture method, and then the immunohistochemical method was used to detect the expression of FSHR receptor in order to identify the purity of granule cels. In the cultured granulosa cels, CD44 and C-Kit expressions were detected immunohistochemicaly, RT-PCR was used to detect ABCG2/Bcrp1, Pou5f1/Oct-4, Nanog gene expressions in ovarian tissue and granulosa cels. RESULTS AND CONCLUSION:Immunohistochemistry detection on paraffin sections showed that a part of ovarian granulosa cels expressed CD34, CD133, ABCG2/Bcrp1 and Pou5f1/Oct-4, and the expression of Pou5f1/Oct-4 protein gradualy increased in the development of ovarian folicles, significantly enhanced during the luteal phase, and then disappeared after the formation of corpus albicans, displaying a periodic expression characteristics. FSHR receptor positive identification rate of primary cels harvested by the foliclar puncture method was more than 95%. Granulosa cels culturedin vitrowere mainly long spindle-shaped or diamond, and some cels presented with aggregation growth and expressed CD44 and C-Kit. RT-PCR test results showed that there were no Nanog in the ovarian tissue and cultured granulosa cels, low expression of Pou5f1/Oct-4 in the ovarian tissue, strong expression of ABCG2/Bcrp1 in the ovarian tissue, weak expression of Pou5f1/Oct-4 in the cultured granulosa cels, and strong expression of ABCG2/Bcrp1 in the cultured granulosa cels. These findings suggest that a part of granulosa cels in the rat ovarian have the characteristics of stem cels.

Objective To investigate the effect of different programs of preconditioning with hyperbaric oxygen (HBO) on spinal cord ischemia-reperfusion injury (I/R) in rabbits. Methods Forty-five New Zealand rabbits aged 4-5 months weighing 2.0-2.5 kg were randomly divided into 5 groups: group S, sham operation ( n = 5);group IR, spinal cord I/R injury (n = 10);group H_(1～3) , the animals were pretreated with 100% O_2 at 2.5 ATA 1 h/d for 5 (group H_1 ), 10 (group H_2 ) , or 20 (group H_3 ) consecutive days respectively 24 h before spinal cord I/R. The animals were anesthetized with iv pentobarbital sodium 30 mg/kg. The artery in the ear and left femoral artery were cannulated for proximal and distal mean blood pressure monitoring. Spinal cord ischemia was produced by cross-clamping of abdominal aorta distal to renal artery for 20 min. Hind-limb motor function was assessed at 48 h after reperfusion according to the modified criteria established by Tarlov (0 = no spontaneous movement, 4= normal motor function) . The animals were then killed and the L_5 segment of the spinal cord was removed for detection of neuronal survival (by HE staining), apoptosis (by TUNEL) and degeneration (by Fluoro-Jade B staining). Results Preconditioning with 5 or 10 d of HBO improved the hind-limb motor function and preserved more normal neurons in the spinal cord after I/R injury. Both apoptotic and degenerative cell death were attenuated in H_1 and H_2 groups. There was no significant difference in hind-limb motor dysfunction and the number of normal neurons in the lumbar spinal cord between H_3 group and I/R group. Conclusion Preconditioning with 5 d or 10 d HBO induces tolerance against spinal cord I/R injury, whereas preconditioning with 20 d of HBO fails to protect the spinal cord from I/R injury.

In this study, the relationship between mycelium morphology and laccase production was studied. The results indicated that the morphology of P. ferulae pellets was changed when glass beads were added. Laccase production showed higher with spherical mycelium than with filamentous or flocculent mycelium. In addition, the spherical mycelium with a diameter of 0.2-0.4 mm highly affected laccase production. Effect of the composition of culture medium on pellets was investigated and results indicated that various concentrations of glucose, corn meal and wheat bran were important to the formation of pellets in diameter of 0.2-0.4 mm. Besides nutrients, the addition of non-nutritional substrates influenced the distribution of P. ferulae pellets. However, the production of laccase was not promoted by non-nutritional substrates.
Culture Media ; Fermentation ; Glass ; chemistry ; Industrial Microbiology ; Laccase ; biosynthesis ; Mycelium ; cytology ; growth & development ; Pleurotus ; cytology ; enzymology

Objective To explore the success rate and the risk of establishment of the acute myocardial infarction model between the beagle dogs and the mini-pigs by interventional technique ,further to provid theoretical basis for choose a more suitable animal model .Methods 6 dogs and 6 mini-pigs were anaesthetized ,then underwent the coronary arteriography via femoral artery .After is-chemic preconditioning the coronary balloon was inflated to occlude the middle left anterior descending coronary for 180 minutes . The electrocardiogram was examined throughout the operation and the pathological sections were examined until the animals were executed one week later .Results All beagle dogs survived ,while 1 case of mini-pigs dead(1/6) .There was 1 cases(1/6) of beagle dogs had acute myocardial infarction ,while 5(5/6)cases in mini-pigs .All mini-pigs had malignant arrhythmia(6/6) but never seen in beagle dogs .The time needed for building a model was similar between the two groups ,the difference had no statistical signifi-cance(P>0 .05) .Conclusion The risk of establish myocardial infarction model in mini-pigs is higher than beagle dogs ,but the suc-cess rate is still high ,it might be the better choice .

To produce recombinant phospholipase A(1) (PLA(1)) by Escherichian coli, the pla gene encoding PLA(1) was amplified from Serratia liquefaciens by PCR and cloned into two vectors pET20-b(+) and pET28-a(+). The two recombinant plasmids were then transformed into E. coli BL21 (DE3) individually to express PLA(1). E. coli BL21(DE3)/pET28a-pla yielded extracellular PLA(1) with an activity of 40.8 U/mL in batch cultivations of shaken flasks by auto-induction, which was accounted for 91% of total enzyme activity. On the basis of primal auto-induction medium, the optimized fermentation medium of PLA(1) contained tryptone 10 g/L, yeast extract 5 g/L, glucose 0.8 g/L, lactose 5 g/L, Na2HPO4 25 mmol/L, KH2PO4 25 mmol/L and 1 mmol/L MgSO4 (final concentration). Glycine (7.5 g/L) was added 6 h after inoculated. After incubated at 37 degrees C for 24 h, extracellular enzyme activity reached 128.7 U/mL.
Cloning, Molecular ; Culture Media ; Escherichia coli ; genetics ; growth & development ; metabolism ; Fermentation ; Lactose ; pharmacology ; Phospholipases A1 ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Serratia liquefaciens ; enzymology

Electrode array misplacement is a rare complication of cochlear implant. This article reports an 11-year-old boy who was mistakenly implanted the cochlear electrode array into the superior semicircular canal during the initial cochlear implant. After the diagnosis was confirmed, he underwent a second cochlear implant and the electrode array were successfully implanted into the cochlea. This article conducted a systematic review of the literature on electrode array misplacement, and the causes of electrode array misplacement were analyzed from different implantation position.
Male ; Humans ; Child ; Electrodes, Implanted ; Reoperation ; Cochlea ; Cochlear Implantation ; Cochlear Implants/adverse effects* ; Semicircular Canals/surgery*

Few tools of gene editing have been developed in Bacillus licheniformis at present. In order to enrich the tools, an FLP/FRT gene editing system that can repeatedly use a single selectable marker was constructed in Bacillus licheniformis, and the system was verified by knocking out an alpha amylase gene (amyL), an protease gene (aprE) and knocking in an exogenous Vitreoscilla hemoglobin gene (vgb). First, knock-out plasmids pNZTT-AFKF of amyL and pNZTT-EFKF of aprE were constructed using thermosensitive plasmid pNZT1 as a carrier. The two knock-out plasmids contained respective homology arms, resistance genes and FRT sites. Then the knock-out plasmids were transformed into Bacillus licheniformis and the target genes were replaced by respective deletion cassette via twice homologous exchange. Finally, an expression plasmid containing FLP recombinase reading frane was introduced and mediated the excision of resistance marker. In order to expand the practicability of the system, knock-in plasmid pNZTK-PFTF-vgb was constructed, with which knock-in of vgb at pflB site was carried out successfully. The results showed that amyL and aprE were successfully knocked out and the marker kanamycin cassette exactly excised. The activities of amylase and protease of deletion mutants were reduced by 95.3% and 80.4% respectively. vgb was successfully knocked in at pflB site and the marker tetracycline cassette excised. The expression of integrated vgb was verified via real-time PCR. It is the first time to construct an FLP/FRT system for gene editing in Bacillus licheniformis, which could provide an effective technical means for genetic modification.
Bacillus licheniformis ; Gene Editing ; Plasmids ; Sequence Deletion

Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.
Flavobacterium ; High-Throughput Screening Assays ; Mirabilis ; Saccharomyces cerevisiae/genetics* ; Tyrosine

Lycopene, as a high value-added terpene compound, has been widely concerned by researchers at home and abroad. Firstly, the ability of lycopene synthesis of Saccharomyces cerevisiae model strains S288c and YPH499 was analyzed and compared. The results showed that YPH499 was more suitable for lycopene synthesis as yeast chassis. Subsequently, the effects of constitutive promoters GPDpr, TEF1pr and inducible promoters GAL1pr, GAL10pr on Lycopene synthesis were compared. The results showed that when GPDpr and TEF1pr were used as promoters of crtE, crtB and crtI in lycopene synthesis pathway, the production of lycopene was 15.31 mg/L after 60 h fermentation in shaking flask. When GAL1pr and GAL10pr were used as promoters, the production was 123.89 mg/L, which was 8.09 times higher. In addition, the methylvaleric acid (MVA) pathway was further modified to overexpress the key enzyme gene of N-terminal truncation, tHMG1 (3-hydroxy-3-methylglutaryl coenzyme A reductase). The lycopene production was 265.68 mg/L, and the yield per cell was 72.79 mg/g. The Saccharomyces cerevisiae strain designed and constructed in this study can express lycopene in high yield per cell, thus could be used in the industrial production of lycopene after further construction and optimization.
Biosynthetic Pathways ; genetics ; Fermentation ; Industrial Microbiology ; Lycopene ; metabolism ; Saccharomyces cerevisiae ; genetics ; metabolism ; Species Specificity