Oncogenic β-catenin-driven liver cancer is susceptible to methotrexate-mediated disruption of nucleotide synthesis / 中华医学杂志(英文版)

BACKGROUND@#Liver cancer is largely resistant to chemotherapy. This study aimed to identify the effective chemotherapeutics for β-catenin-activated liver cancer which is caused by gain-of-function mutation of catenin beta 1 ( CTNNB1 ), the most frequently altered proto-oncogene in hepatic neoplasms.@*METHODS@#Constitutive β-catenin-activated mouse embryonic fibroblasts (MEFs) were established by deleting exon 3 ( β-catenin Δ(ex3)/+ ), the most common mutation site in CTNNB1 gene. A screening of 12 widely used chemotherapy drugs was conducted for the ones that selectively inhibited β-catenin Δ(ex3)/+ but not for wild-type MEFs. Untargeted metabolomics was carried out to examine the alterations of metabolites in nucleotide synthesis. The efficacy and selectivity of methotrexate (MTX) on β-catenin-activated human liver cancer cells were determined in vitro . Immuno-deficient nude mice subcutaneously inoculated with β-catenin wild-type or mutant liver cancer cells and hepatitis B virus ( HBV ); β-catenin lox(ex3)/+ mice were used, respectively, to evaluate the efficacy of MTX in the treatment of β-catenin mutant liver cancer.@*RESULTS@#MTX was identified and validated as a preferential agent against the proliferation and tumor formation of β-catenin-activated cells. Boosted nucleotide synthesis was the major metabolic aberration in β-catenin-active cells, and this alteration was also the target of MTX. Moreover, MTX abrogated hepatocarcinogenesis of HBV ; β-catenin lox(ex3)/+ mice, which stimulated concurrent Ctnnb1- activated mutation and HBV infection in liver cancer.@*CONCLUSION@#MTX is a promising chemotherapeutic agent for β-catenin hyperactive liver cancer. Since repurposing MTX has the advantages of lower risk, shorter timelines, and less investment in drug discovery and development, a clinical trial is warranted to test its efficacy in the treatment of β-catenin mutant liver cancer.

Study on the Anti-inflammatory Activity and Mechanism of Different Components from Codonopsis Radix Polysaccharides / 中国药房

OBJECTIVE：To screen the components with better anti-inflammatory effect from Codonopsis Radix polysaccharide and explore the anti-inflammatory mechanism. METHODS ：Using Codonopsis Radix as sample ，the crude polysaccharide （CP） was obtained by water extraction and alcohol precipitation. After CP was separated and purified by Fast Flow DEAE Sepharose ， SephadexG-150 gel column and so on ，the components CP 1-2-1 and CP 3-1-1 were obtained and then characterized. The survival rate of RAW 264.7 cell was determined by MTT method after cultured with CP ，CP1-2-1 and CP 3-1-1（0.01，0.1，1，10，100 μg/mL）for 24 and 48 h respectively. The cells were divided into blank group （blank culture medium ），model group （1 μg/mL LPS）and low-，medium- and high-concentration groups of 3 kinds of Codonopsis Radix polysaccharide （1 μg/mL LPS+25，50， 100 μg/mL CP，CP1-2-1，CP3-1-1 solution）. After cultured for 24 h，NO content in cell culture solution ，mRNA expressions of TLR4，IL-6，NF-κB and TNF-α in cells were determined. RESULTS：The sugar content of CP reached （92.20±0.73）％. CP 1-2-1 was a single neutral polysaccharide composed of fructose with a relative molecular weight of 25.8 kDa. CP 3-1-1 was an acidic heteropolysaccharide composed of arabinose ，rhamnose，galactose and galacturonic acid and so on ，with a relative molecular weight of 49.5 kDa. Cell survival rates were higher than 99％，after cultured with 3 kinds of polysaccharide for 24 and 48 h. Compared with blank group ，NO content in cell culture solution and mRNA expressions of TLR 4，IL-6，NF-κB and TNF-α in cells were increased significantly in model group （P＜0.05 or P＜ . Compared with model group ， NO content in cell 2016CFB412） culture solution of each administration group was significantly reduced，mRNA expressions of TLR 4，NF-κB，TNF-α，IL-6 in cells in 50，100 μg/mL CP group were significantly decreased ong1991@163.com （P＜0.05 or P＜0.01）；mRNA expressi ons of TLR 4，NF-κB in cells in 25 µg/mL CP 1-2-1 group and mRNA expressions of TLR 4，NF-κB，TNF-α，IL-6 in cells in 50，100 µg/mL CP 1-2-1 group were significantly decreased （P＜0.05 or P＜0.01）；mRNA expressionsof IL- 6 in cells in 25 µg/mL CP 3-1-1 group，mRNA expressions of NF-κB，TNF-α，IL-6 in cells in 50 µg/mL CP 3-1-1 group and mRNA expressions of TLR 4，NF-κB，TNF-α，IL-6 in cells in 100 µg/mL CP 3-1-1 group were significantly decreased （P＜0.05 or P＜0.01）. CONCLUSIONS ：CP，CP 1-2-1 and CP 3-1-1 all have certain anti-inflammatory activities. The mechanism may be related to inhibiting the generation and releasing of inflammatory cytokines such as TNF-α，IL-6 by inhibiting the activation of TLR 4/NF-κB pathway.

Study on Quality Standard Improvement of Pheretima / 中国药房

OBJECTIVE： To provide reference for improving the quality standard of Pheretima. METHODS： The contents of hypoxanthine and inosine in medicinal material samples were determined by HPLC. HPLC fingerprint of Pheretima was established according to “Similarity evaluation system for TCM chromatogramtic fingerprint” （2012 edition） software， and similarity evaluation was conducted. The determination was performed on Purospher STAR RP-18 endcapped with mobile phase consisted of methanol-water （gradient elution） at the flow rate of 1 mL/min. The detection wavelength was 248 nm， and the column temperature was set at 30 ℃. The sample size was 20 μL. RESULTS： The results of methodological investigation of content determination showed that the linear range of hypoxanthine and inosine were 1.58-31.6 μg/mL （r＝0.999 9）， 5.52-110.4 μg/mL（r＝0.999 8）， respectively. limits of quantify were 0.316， 0.552 μg/mL， respectively； limits of detection were 0.158， 0.110 μg/mL， respectively； RSDs of precision， stability （24 h） and repeatability tests were all less than 2.0％ （n＝6）. Average recovery rates were 103.0％ （RSD＝1.7％， n＝6） and 101.2％ （RSD＝1.2％， n＝6）， respectively. HPLC fingerprint for 15 batches of samples were established， and 8 common peaks were identified. The similarity of HPLC fingerprint of 14 batches of sample with control fingerprint R was higher than 0.900. CONCLUSIONS： The established method for content determination of hypoxanthine and inosine and HPLC fingerprint of Pheretima are simple， accurate and reproducible， and can be used for quality control of Pheretima.

Quality Standard Improvement for Angelica Sinensis / 中国药师

Objective: To establish and optimize the HPLC fingerprints of Angelica sinensis medicinal material and determine the ligustilide content to improve the quality standard for Angelica sinensis and improve the quality control level of Chinese angelica medici-nal material and preparations. Methods: A method for the determination of ligustilide was optimized by HPLC. The column was eluted on an Agilent ZORBAX SB C18(250 mm×4. 6 mm, 5 μm) column with acetonitrile-water (60 ∶ 40) as the mobile phase. The flow rate was 1. 0 ml·min-1, the detection wavelength was 326 nm, and the column temperature was at 35 ℃. The HPLC fingerprints of 13 batches of Angelica sinensis from different origins and methodological investigations were established and validated to set up an HPLC fingerprinting evaluation method for Angelica sinensis. Acetonitrile-0. 1% formic acid was used as the mobile phase with gradient elu-tion. The flow rate was 1. 0 ml·min-1, the detection wavelength was 280nm, and the column temperature was at 25℃. Results: The results showed that under the above HPLC conditions, ligustilide had good linearity within the range of 0. 032 3-0. 645 5 mg·ml-1(r=0. 999 9), and the average recovery was 100. 5% (RSD=1. 61% ,n=6). The quality fraction of ligustilide in Angelica sinensis was 0. 885 6%-2. 382 2% . Through the establishment of HPLC fingerprints of Angelica sinensis, the characteristic profiles with better peak shape and degree of separation and 18 common peaks with better resolution were obtained. The similarities of the 13 batches of angelica were all between 0. 9 and 1. 0. Conclusion: According to the methodological investigation, the HPLC fingerprints and ligustilide con-tent determination method of Angelica sinensis are simple, reliable, stable and feasible.

Improvement of inhibitors tolerance of Saccharomyces cerevisiae by overexpressing of long chain sphingoid kinases encoding gene LCB4 / 生物工程学报

By-products released from pretreatment process of lignocellulose seriously hinder the development of cellulosic fuel ethanol. Therefore, the great way to increase the efficiency of cellulosic ethanol production is improvement of Saccharomyces cerevisiae tolerance to these inhibitors. In this work, the effects of LCB4 gene overexpression on cell growth and ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses were studied. Compared to the control strain S288C-HO, the recombinant strain S288C-LCB4 grew better on YPD solid medium containing 10 g/L acetic acid, 1.5 g/L furfural and 1 g/L vanillin. Ethanol yields of recombinant strain S288C-LCB4 were 0.85 g/(L·h), 0.76 g/(L·h) and 1.12 g/(L·h) when 10 g/L acetic acid, 3 g/L furfural and 2 g/L vanillin were supplemented into the fermentation medium respectively, which increased by 34.9%, 85.4% and 330.8% than the control strain S288C-HO. Meanwhile, ethanol fermentation time was reduced by 30 h and 44 h under furfural and vanillin stresses respectively. Further metabolites analysis in fermentation broth showed that the recombinant strain produced more protective compounds, such as glycerol, trehalose and succinic acid, than the control strain, which could be the reason for enhancing strain tolerance to these inhibitors from pretreatment process of lignocellulose. The results indicated that overexpression of LCB4 gene could significantly improve ethanol fermentation in S. cerevisiae S288C under acetic acid, furfural and vanillin stresses.

Single-domain antibody - advances in research and application / 生物工程学报

Conventional IgG is composed of heavy and light chains. The light chain has one variable region (VL) and one constant region (CL) domain, whereas the heavy chain has one variable region (VH) and three constant region domains (CH1, CH2 and CH3). Single domain antibody (sdAb) is a kind of antibody that is composed of a variable domain of heavy chain and devoid of the light chain completely. Due to its small size, it is also called as nanobody. Although the sdAb has a simple structure, it can exhibit a comparable even better antigen-binding affinity than conventional antibody. Compared with conventional antibody, the small size, high stability and simplicity in recombinant expression are representative advantages of sdAb. In recent years, scientists are becoming increasingly interested in the roles of sdAb in fundamental biomedical research and clinical application. In this review, we summarized the structural features, physicochemical properties, screening strategies and recent advances in application of sdAb.

Effect of Meicha Protein on the Heart of Spontaneously Hypertensive Rats / 医药导报

Objective To observe the protective effect of meicha protein on the heart of spontaneously hypertensive rats(SHR),and explore its mechanism.Methods Fourty healthy SHR rats were randomly divided into 4 groups:model control group,Meicha protein low dose group(70 mg·kg-1)、Meicha protein high dose group(140 mg·kg-1),Compound Kendir Leaves Tablets group(50 mg·kg-1),n=10.The rats were orally administered twice daily by gavage for seven weeks,measuring blood pressure in each group fort nightly.1 h after the last administration,drawing off the blood from carotid,stripping off the heart tissue,and the organ index was calculated;Taking a part of the tissue with 4% paraformaldehyde for Pathological histology.Detection of serum NO,ET-1 levels as well as the organization of the ACE and Ang II mRNA expression to explore the mechanism of its buck.Results Meicha protein could significantly reduce the blood pressure of SHR;The impact on the rat organ coefficient was not obvious,but had a protective effect on heart tissue.Compared with the model control group,the contents of NO an ET-1 were significantly increased(P<0.01).Compared with the model group,the high dose of Meicha protein could induce ACE,AngⅡ,CYP11B2.The expression of mRNA was significantly decreased(P<0.01).Conclusion The possible mechanism of Meicha protein antihypertensionis relevant to increase the content of NO in serum,reduce the content of ET-1 in serum,reduce mRNA expression of ACE and AngⅡin cardiac tissue.

Study on Quality Standard of Huangdi Cataplasm / 医药导报

Objective To develop the quality standard for evaluating Huangdi cataplasm. Methods Thin layer chromatography (TLC) was used to qualitatively identify Astragalus membranaceus (Fisch.) Bunge,Rheum palmatum Linn,Rhizoma Chuanxiong,Angelica sinensis and Resina Draconis in Huangdi cataplasm.HPLC method was used to determine astragaloside A and loureirin B in Huangdi cataplasm. Results The Astragalus membranaceus (Fisch.) Bunge,Rheum palmatum Linn,Rhizoma Chuanxiong,Angelica sinensis and Resina Draconis were well separated by TLC without interference in the negative control.content of Astragaloside A and loureirin B showed good liner relationships with respective peak area within the range of 6.96-23.2 μg,and 0.072-0.648 μg,with r = 0.999 5,r = 0.999 9, respectively;and the average recovery was 97.18%,and 96.93%,RSD was 1.21%(n= 6),1.53% (n = 6 ), respectively. Conclusion The established qualitative and quantitative detection method is simple, specific, reproducible, accurate and reliable, which can be used for quality control of Huangdi cataplasm.

On-line detecting of transformer winding deformation based on parameter identification of leakage inductance / 药物分析学报

Transformers are required to demonstrate the ability to withstand short circuit currents. Over currents caused by short circuit can give rise to windings deformation. In this paper, a novel method is proposed to monitor the state of transformer windings, which is achieved through on-line detecting the leakage inductance of the windings. Specifically, the mathematical model is established for on-line identifying the leakage inductance of the windings by applying least square algorithm (LSA) to the equivalent circuit equations. The effect of measurement and model inaccuracy on the identification error is analyzed, and the corrected model is also given to decrease these adverse effect on the results. Finally, dynamic test is carried out to verify our method. The test results clearly show that our method is very accurate even under the fluctuation of load or power factor. Therefore, our method can be effectively used to on-line detect the windings deformation.

MORPHOMETRIC ANALYSIS ON THE MITOCHONDRIA OF MYOCARDIUM IN EXERCISE TRAINED RATS / 中国运动医学杂志

Adult male rats were divided at random into two groups and trained on the treadmill running schedules, 60 min/day and 10min/day for group A and B separately. Four weeks later the ultrastructure of the left ventricular myocardium was examined and the specific surface of mitochondria wasmeasured. Comparing to the corresponding control group, the specific surface of mitochondria in the two groups of trained rats was decreased significantly. (5.883+0.12 um and 6.050?0.12 um to 6.564?0.17 um). This means myocardium mitochondria in trained rats were swelling. The reason might be that the myocardium injury was induced by exercise training.

THE EFFECT OF CGRP ON THE EXPRESSION OF CREB mRNA IN RAT HIPPOCAMPUS AND PARIETAL CORTEX DURING FOCAL CEREBRAL ISCHEMIA AND REPERFUSION / 解剖学报

Objective To investigate the effect of calcitonin gene related peptide(CGRP) on the expression of cyclic AMP response element binding protein(CREB) mRNA in rat hippocampus and parietal cortex during focal cerebral ischemia and reperfusion(I/R). Methods Focal cerebral ischemia/reperfusion model was induced by occluding of the right middle cerebral artery using the intraluminal suture method.Hybridization in situ experiment was used to detect the expression of CREB mRNA in the ipsilateral hippocampal CA1 region and parietal cortex during different reperfusion periods.The positive product of CREB mRNA was analyzed by image analysis system. Results There was a distinct expression of CREB mRNA in right hippocampal CA1 region and parietal cortex in sham group.The absorbency of CREB mRNA positive product reduced in I/R group as compared to sham group,while it increased in CGRP group than I/R group(P