Cloning and expression analysis of U6 promoters in Panax quinquefolius / 中国中药杂志

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Effect of Curdione on MDA-MB-231 Cell Cycle and Apoptosis / 中国实验方剂学杂志

Objective:To investigate the effects of curdione on the proliferation， apoptosis and cell cycle of triple negative breast cancer cell line MDA-MB-231. Method:MDA-MB-231 cells were cultured<italic> in vitro</italic> with capecitabine （positive control） and curdione at different concentrations （125， 250， 500， 1 000， and 2 000 μmol·L^-1）， respectively， for detecting their viability using the cell counting kit-8 （CCK-8） at 24 and 48 h. Three effective inhibitory concentrations （250， 500， and 1 000 μmol·L^-1） against cell proliferation were selected for subsequent experiments. The effect of curdione on cell cycle was determined by flow cytometry combined with propidium iodide （PI） staining. After the set-up of high-concentration （2 000 μmol·L^-1） group， the effect of curdione on cell mitochondrial membrane potential was measured by JC-1（5，5，6，6-tetrachloro-1，1，3，3-tetraethylbenzimidazolylcarbocyanine iodide） staining， followed by the detection of cell apoptosis by flow cytometry combined with Annexin V-FITC/PI double staining. The changes in cell cycle status and apoptosis-related protein expression following curdione intervention were assayed by Western blot. Result:Compared with the blank control， curdione at 250， 500， 1 000， and 2 000 μmol·L^-1significantly inhibited the proliferation of MDA-MB-231 cells （<italic>P</italic><0.01）， exhibiting a concentration- and time-response relationship. The half maximal inhibitory concentration （IC₅₀） values at 24 and 48 h were 1 607 and 1 401 μmol·L^-1， respectively. Curdione at 250， 500， and 1 000 μmol·L^-1 arrested cells in G₁ phase. Curdione at 250 μmol·L^-1had no effect on cell mitochondrial membrane potential， which， however， declined significantly in the 500， 1 000， and 2 000 μmol·L^-1groups （<italic>P</italic><0.05， <italic>P</italic><0.01）. Curdione at 250， 500， and 1 000 μmol·L^-1obviously increased the proportion of apoptotic cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. Curdione at each concentration elevated the Bcl-2-associated X protein （Bax）/B-cell lymphoma 2 （Bcl-2） ratio （<italic>P</italic><0.05， <italic>P</italic><0.01）， but did not change the cysteinyl aspartate-specific protease-3 （Caspase-3） expression. The protein expression levels of Caspase-9， cleaved Caspase-9， cleaved Caspase-3， p53， and p21 were up-regulated （<italic>P</italic><0.05）. Conclusion:A certain concentration of curdione inhibits the proliferation of MDA-MB-231 cells， which may be related to its efficacy in arresting cell cycle and inducing apoptosis.

Effect and Mechanism of Curdione on Migration and Invasion of Breast Cancer HCC1937 Cells / 中国实验方剂学杂志

Objective: To investigate effect of curdione on the migration and invasion of human breast cancer HCC1937 cells and its mechanism.Method: HCC1937 cells were cultured in vitro and treated with curdione at various doses (0, 12.5, 25, 50, 100, 200, 400 μmol·L-1) for 24, 48 h, the cell viability was detected by cell counting kit-8 method. curdione groups (12.5, 25, 50 μmol·L-1) and blank group were established. The effect of curdione on the adhesion of HCC1937 cells was detected by the cell adhesion assay. The effect of curdione on migration of HCC1937 cells was detected by wound healing assay. The effect of curdione on the migration and invasion of HCC1937 cells were detected by transwell chamber assay. The effect of curdione on regulation of mitogen-activated protein kinase(MAPK)and protein kinase B(Akt)signaling pathways and the protein expressions of matrix metalloproteinases-2 (MMP-2) and matrix metalloproteinases-9 (MMP-9) of HCC1937 cells were detected by the Western blot analysis. Effect of curdione on mRNA expressions of MMP-2 and MMP-9 of HCC1937 cells were detected by Real-time PCR.Result: Compared with the blank group, curdione (12.5, 25, 50 μmol·L-1) groups had no significant effect on cell viability, but a remarkable effect on cell viability HCC1937 cells, and cell viability was gradually decreased with the increase of the concentration of curdione (PP-1) had a significant effect on cell adhesion rate, migration rate and invasion rate of HCC1937 cells (PP-1) could down-regulate phosphorylation levels of key proteins extracellular regulated protein kinases(ERK), c-Jun N-terminal kinase(JNK), Akt on MAPK and Akt signaling pathways (PConclusion: curdione can inhibit the migration and invasion of human breast cancer HCC1937 cells, and the mechanism may be related to down-regulation of phosphorylation levels of key proteins ERK, JNK, Akt on MAPK and Akt signaling pathways, so as to reduce the expressions of MMP2 and MMP-9.

Determination of iridoid glycosides in Lonicerae Japonicae Flos and its patent medicine Lianhua Qingwen Capsule by dispersive solid phase extraction-HPLC / 中草药

Objective: To develop a new method based on dispersive solid phase extraction-high performance liquid chromatography (DSPE-HPLC) for the rapid extraction, cleansing, and determination of iridoid glycosides (loganic acid, morroniside, loganin, sweroside, secoxyloganin, and secologain dimethyl acetal) in Lonicerae Japonicae Flos (LJF) and the related traditional Chinese patent medicine (TCPM) Lianhua Qingwen Capsule (LQC). Methods: Ultrasonic extraction was used for the extraction, different purification material was investigated for the rapid purification, and HPLC method was used for the determination of the content of iridoid glycosides. Results: The purification effect of LJF extract was much better by using diatomite as the purification material; The purification of LQC extract was much better by using diatomite-active carbon 80∶20. This method was simple and rapid, and has high efficiency. It could be used for the extraction, cleaning, and determination of iridoid glycosides in LJF and related prescription combined with HPLC. Conclusion: This study expands the application of DSPE in the extraction and purification of complex matrix traditional Chinese material medicine (TCMM) and TCPM, and supplies the new method and data support for the extraction and purification of bioactive compounds from complex matrix TCMM and TCPM.

Traditional Chinese medicinal herbs combined with epidermal growth factor receptor tyrosine kinase inhibitor for advanced non-small cell lung cancer: a systematic review and meta-analysis / 中西医结合学报

<p><b>BACKGROUND</b>Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) targeted treatment has been a standard therapy for advanced non-small cell lung cancer (NSCLC), but it is not tolerated well by all patients. In China, some studies have reported that traditional Chinese medicinal herbs (TCMHs) may increase efficacy and reduce toxicity when combined with EGFR-TKI, but outside of China few studies of this kind have been attempted.</p><p><b>OBJECTIVE</b>This study is intended to systematically review the existing clinical evidence on TCMHs combined with EGFR-TKI for treatment of advanced NSCLC.</p><p><b>SEARCH STRATEGY</b>PubMed, the Cochrane Library, the Excerpta Medica Database (EMBASE), the China BioMedical Literature (CBM), and the China National Knowledge Infrastructure (CNKI) and web site of the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the World Conference of Lung Cancer (WCLC) were searched; the search included all documents published in English or Chinese before October 2013.</p><p><b>INCLUSION CRITERIA</b>We selected randomized controlled trials based on specific criteria, the most important of which was that a TCMH plus EGFR-TKI treatment group was compared with an EGFR-TKI control group in patients with advanced NSCLC.</p><p><b>DATA EXTRACTION AND ANALYSIS</b>The modified Jadad scale was used to assess the quality of studies. For each included study, patient characteristics, treatment details, therapeutic approach and clinical outcomes were collected on a standardized form. When disagreements on study inclusion or data extracted from a study emerged, the consensus of all coauthors provided the resolution. The clinical outcome metrics consisted of objective response rate (ORR; complete response + partial response divided by the total number of patients), disease control rate (DCR; complete response + partial response + no change divided by the total number of patients), survival rate, improved or stabilized Karnofsky performance status (KPS), and severe toxicity. RevMan 5.0 software was used for data syntheses and analyses. Risk ratio (RR) and 95% confidence interval (CI) were calculated; if the hypothesis of homogeneity was not rejected (P>0.1, I(2)<50%), the fixed-effect model was used to calculate the summary RR and the 95% CI. Otherwise, a random-effect model was used.</p><p><b>RESULTS</b>In this review, 19 studies were included based on the selection criteria. Of them, 13 studies were of high quality and 6 studies were of low quality, according to the modified Jadad scale. When the TCMH plus EGFR-TKI treatment groups were compared with the EGFR-TKI control groups the meta-analysis demonstrated a statistically significant higher ORR (RR 1.34; 95% CI 1.15 to 1.57; P=0.000 2), DCR (RR 1.18; 95% CI 1.09 to 1.27; P<0.000 1), one-year survival rate (RR 1.21; 95% CI 1.01 to 1.44; P=0.04), 2-year survival rate (RR 1.91; 95% CI 1.26 to 2.89; P=0.002) and improved or stable KPS (RR 1.38; 95% CI 1.26 to 1.51; P<0.000 01). Severe toxicity for rash was decreased (RR 0.55; 95% CI 0.32 to 0.94; P=0.03), as were nausea and vomiting (RR 0.17; 95% CI 0.04 to 0.72; P=0.02) and diarrhea (RR 0.46; 95% CI 0.24 to 0.89; P=0.02). Sensitivity analysis indicated that findings of the meta-analysis were robust to study quality. In the funnel plot analysis, asymmetry was observed, and publication bias was indicated by Egger's test (P=0.03).</p><p><b>CONCLUSION</b>TCMH intervention can increase efficacy and reduce toxicity when combined with EGFR-TKI for advanced NSCLC, although this result requires further verification by more well designed studies.</p>

Sequence analysis of varicella-zoster virus gE gene in varicella-zoster virus strains with different clades / 病毒学报

To analyze the gE gene sequence of varicella-zoster virus (VZV) strains of different clades and subclades currently circulating in China. Eighteen skin lesion fluid swabs or skin scab pieces from patients with chickenpox or shingles were obtained from Beijing, Changchun, Lhasa and Urumqi between December 2010 and June 2011. The genotype of the virus strains was determined by a group of single nucleotide polymorphism (SNP) located in 15 ORFs, and the full-length gE genes of 18 strains representing all the clades in the study was amplified by PCR and sequenced. In addition to the synonymous mutations and non-synonymous mutations that were reported in the literature, there were 3 novel non-synonymous mutations (C56T, C1109T, C917A) and 4 new synonymous mutations (C54T, T1075C, T816C, G279A) found in the 8 strains analyzed. We found the VZV strains of clade 5 in Xinjiang for the first time,and the genotypes of some VZV strains circulating in Chagnchun could not be determined by the present methods. The analysis of gE gene sequences,revealed a novel non-synonymous mutations in the e1 and c1 epitopes, corresponding to the amino acid change of serine to tyrosine.

Xuefu zhuyu oral liquid intervened stress-stimulated depression model rats / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe effects of xuefu zhuyu Oral Liquid (XZOL) on the brain behavior and monoamine neurotransmitter 5-HT, and brain derived neurotrophic factor (BDNF) content on depression model rats.</p><p><b>METHODS</b>Male SD rats were randomly divided into the control group, the model group, the XZOL group, and the Deanxit Tablet group, 12 in each group. The depressive rat model was established by chronic unpredictable mild stress method. XZOL was administered to rats in the XZOL group by gastro-gavage, while Deanxit Tablet was given to those in the Deanxit Tablet group by gastrogavage. The intervention lasted for two weeks. The behavioral changes were observed by sucrose water consumption test and open-field test. The 5-HT and BDNF contents were detected using ELISA.</p><p><b>RESULTS</b>After chronic stress stimulus, experimental rats in the model group might have abnormal behavioral changes and lowered 5-HT content, showing statistical difference when compared with the control group (P <0.01). No obvious change in stimulated rats' behavior after intervention of XZOL and Deanxit Tablet. 5-HT content was not obviously reduced (P>0.05). Besides, XZOL was superior to Deanxit Tablet in increasing the 5-HT content (P<0.05). But the brain BDNF level of rats in the model group was not statistically different from that of rats in the model group (P >0.05), while the brain BDNF level of rats in the XZOL group and the Deanxit Tablet group was lower than that of rats in the model group (P <0.01).</p><p><b>CONCLUSIONS</b>Stress can lead to behavioral changes and lowered 5-HT content of rats. The intervention of XZOL could fight against depression-induced behavioral changes and increase 5-HT content. But it did not significantly affect the brain BDNF level. We inferred that it might not effect through the BDNF pathway.</p>

The genotyping and molecular evolution of varicella-zoster virus / 病毒学报

Varicella-zoster virus (VZV, Human herpesvirus 3) is a member of the family Herpesviridae, and is classified as alpha-subfamily along with HSV-1 and HSV-2. VZV is the causative agent of chicken pox (varicella) mostly in children, after which it establishes latency in the sensory ganglia with the potential to reactivate at a later time to cause shingles (zoster). Increasing molecular epidemiological studies in recent years have been performed to monitor the mutations in VZV genome, discriminate vaccine virus from wild type virus, study the phylogeny of VZV strains throughout the world, and understand the evolution of the different clades of VZV. The progress has great impact on the fields of epidemiology, virology and bioinformatics. In this review, the currently available data concerning the geographic distribution and molecular evolution of VZV clades are discussed.

Effects of qisheng mixture on chemotherapy induced myelosuppression in patients with colorectal cancer / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To observe the intervention of Qisheng Mixture (QM) on the chemotherapy induced myelosuppression in patients with colorectal cancer.</p><p><b>METHODS</b>One hundred and twenty patients with colorectal cancer at Ruijin Hospital, Shanghai Jiaotong University School of Medicine were randomly assigned to the pure chemotherapy group (as the control group) and the QM + chemotherapy group (as the treatment group), 60 in each group. All patients received FOLFOX4 or XELOX regimen for totally 6 cycles. Patients in the treatment group took QM 150 mL at the end of chemotherapy, once in the morning and once in the evening for 7 successive days, totally 6 therapeutic courses. The total and average dosages of using granulocyte colony stimulating factor (G-CSF) were observed in all patients. The changes of white blood cell (WBC) counts were determined before chemotherapy and after the 6th chemotherapy. The hemoglobin (Hb), red blood cell (RBC), and platelet (PLT) counts were observed before chemotherapy, before the 4th chemotheray, and after the 6th chemotherapy. The clinical symptoms integrals (fatigue, liability to catch cold, aphthous stomatitis, pharyngalgia, pale complexion, poor appetite, vomiting, diarrhea, and so on) and the safety indicators (the functions of the liver and kidney, urine routines) were observed. The grading toxic and adverse reactions, KPS scoring, body weight, and the efficacy of the symptoms integrals were compared between the two groups.</p><p><b>RESULTS</b>During the treatment period the total and average dosages of G-CSF used were larger in the control group than in the treatment group (P<0.01). After treatment the WBC count of the two groups were reduced with statistical difference (P<0.01). The WBC counts were higher in the treatment group than in the control group in the whole therapeutic process except the first chemotherapy (P<0.01, P<0.05). Compared with before treatment in the same group, RBC and PLT were reduced in the two groups before the 4th chemotherapy, RBC, Hb, and PLT were reduced after treatment (P<0.05, P<0.01). Better effects on body weight were obtained in the treatment group than in the control group with statistical difference (P<0.01). Compared with the control group, the clinical symptoms integrals such as fatigue, liability to catch cold, pharyngalgia, pale complexion, poor appetite, vomiting, and diarrhea were reduced (P<0.01). Compared with the control group, the toxic and adverse reactions were reduced in the treatment group before the 4th chemotherapy (P<0.01).</p><p><b>CONCLUSIONS</b>QM could effectively intervene chemotherapy induced myelosuppression in patients with colorectal cancer. It was a safe Chinese medicine compound with lower toxicity.</p>

A murine model with murine cytomegalovirus infection resulting in colon inflammation after allogeneic skin transplantation / 病毒学报

<p><b>UNLABELLED</b>To provide a reliable animal model for study of human CMV disease in gastrointestinal track, we tried to infect with murine cytomegalovirus (MCMV) in mice that were received allogenetic skin transplantation under immunosuppression. (1) Skin transplantation was performed between 18 donor C57BL/6 mice and 72 recipient BALB/c mice. (2) All recipient mice were then given Cyclosporine at 12 mg/kg daily for 2 weeks by intraperitoneal injection. Mice were randomly divided into 3 groups. Two experimental groups were received MCMV-infected mouse embryonic fibroblasts (MEF) at 10(4) PFU and 10(5) PFU respectively, and the control group received MEF only. We observed any possibly pathophysiological behavior changes and recorded the changes in body weight. The mice were sacrificed at 5d, 9d, 14d, 21d post infection and colon tissue was collected for analysis.</p><p><b>RESULTS</b>Mice infected with MCMV at 10(5) PFU group showed anorexia, lethargy and degression in locomotor activity. This group of mice showed significant decrease in body weight than that of other groups. Colon tissues were collected 14 days after infection. Histological examination revealed that the mucous layer became thinner in the proximal colon and increased number of lymphoid follicles in distal colon in infected animals. The changes in the mucosal structure was most prominent in the group 10(5) PFU MCMV. Viral DNA was present in the colon by in situ hybridization for IE1 gene, and viral gB transcript was positive by RT-PCR. One of the viral major proteins, pp65, was widely distributed in the colon by immunohistochemistry. These data demonstrated that MCMV established infection in colon of the mice after allogenetic skin transplantation. Electron microscopy showed that there were herpes virus particles in the colon tissue.</p><p><b>CONCLUSION</b>Infection with MCMV in mouse after allogenetic skin transplantation by nasal cavity inoculation resulted in the pathological changes in colon tissue similar to that of inflammation in human colon. The small animal model of colon inflammation may provide a platform for further study of pathogenesis as well as medical intervention of HCMV involved inflammation of human bowel.</p>

Genetic analysis of clinical varicella-zoster virus isolates collected in China / 中华流行病学杂志

Objective To study the genetic characteristrics of vaccine and varicella-zoster virus(VZV)strains isolated from patients with chickenpox or zoster by molecular analysis.Methods SNP based VZV genetic characteristrics were analyzed in 19 VZV isolates using the restriction fragment length polymorphisms analysis of DNA fragments of the open reading frames 6,38,62 and sequence alignment of the open reading frames 1,31,51,62.Results All vaccine strains were revealed Alu Ⅰ~-Pst Ⅰ~-Sma Ⅰ~+ BssH Ⅱ~+ Nae Ⅰ~+,96% clinical isolates were revealed Alu Ⅰ~+ Pst Ⅰ ~+Sma Ⅰ BssHⅡ Nae Ⅰ~-,2% clinical isolates were revealed Alu Ⅰ~-Pst Ⅰ~-Sma Ⅰ~+ BssH Ⅱ~+,2%clinical isolates were revealed Alu Ⅰ~+ Pst Ⅰ~-Sma Ⅰ~-BssHⅡ Nae Ⅰ~-by restriction fragment length polymorphisms analysis and sequence alignment revealed the mutations also presented in this four vaccine strains.Conclusion Use the restriction fragment length polymorphisms analysis of DNA fragments of the open reading,frames 6 and 62 could be distinguished VZV wild-type strains and vaccine strain in clinical isolates in China.In order to find the adverse effect caused by vaccine from certain company's,analysis on the SNPs in ORFs 1,31,51 and 62 is needed.