Cloning and expression analysis of ANR genes from different species of Lonicera japonica Thunb / 药学学报

Anthocyanidin reductase (ANR) is one of the key enzyme in the flavonoid biosynthetic pathway, and its catalytic activity is important for the synthesis of plant anthocyanin. In this study, specific primers were designed according to the transcriptome data of Lonicera japonica Thunb., and the CDS, gDNA and promoter sequences of ANR genes from Lonicera japonica Thunb. and Lonicera japonica Thunb. var. chinensis (Wats.) Bak. were cloned. The results showed that the CDS sequences of LjANR and rLjANR were 1 002 bp, the gDNA sequences were 2 017 and 2 026 bp respectively, and the promoter sequences were 1 170 and 1 164 bp respectively. LjANR and rLjANR both contain 6 exons and 5 introns, which have the same length of exons and large differences in introns. The promoter sequences both contain a large number of light response, hormone response and abiotic stress response elements. Bioinformatics analysis showed that both LjANR and rLjANR encoded 333 amino acids and were predicted to be stable hydrophobic proteins without transmembrane segments and signal peptides. The secondary structures of LjANR and rLjANR were predicted to be mainly consisted of α-helix and random coil. Sequence alignment and phylogenetic analysis showed that LjANR and rLjANR had high homology with Actinidia chinensis var. chinensis, Camellia sinensis and Camellia oleifera, and were closely related to them. The expression levels of LjANR and rLjANR were the highest in flower buds and the lowest in roots. The expression patterns at different flowering stages were similar, with higher expression levels in S1 and S2 stages and then gradually decreased until reaching the lowest level in S4 stage, after a slow increase in S5 stage, the expression levels decreased again. The expression levels of ANR genes in the two varieties showed significant differences in roots, S2 and S5 stages, while the differences in stems, flower buds, S1, S3 and S6 stages were extremely significant. The prokaryotic expression vector pET-32a-LjANR was constructed for protein expression. The target protein was successfully expressed of about 59 kD. This study lays a foundation for further study on the function of ANR gene and provides theoretical guidance for breeding new varieties of Lonicera japonica Thunb.

Comparison of effectiveness between unilateral biportal endoscopic lumbar interbody fusion and endoscopic transforaminal lumbar interbody fusion for lumbar spinal stenosis combined with intervertebral disc herniation / 中国修复重建外科杂志

OBJECTIVE@#To compare the effectiveness between unilateral biportal endoscopic lumbar interbody fusion (ULIF) and endoscopic transforaminal lumbar interbody fusion (Endo-TLIF) in treatment of lumbar spinal stenosis combined with intervertebral disc herniation.@*METHODS@#A clinical data of 64 patients with lumbar spinal stenosis and intervertebral disc herniation, who were admitted between April 2020 and November 2021 and met the selection criteria, was retrospectively analyzed. Among them, 30 patients were treated with ULIF (ULIF group) and 34 patients with Endo-TLIF (Endo-TLIF group). There was no significant difference in baseline data such as gender, age, disease duration, lesion segment, preoperative visual analogue scale (VAS) score of low back pain and leg pain, Oswestry disability index (ODI), spinal canal area, and intervertebral space height between the two groups ( P>0.05). The operation time, intraoperative blood loss, hospital stays, and postoperative complications were compared between the two groups, as well as the VAS scores of low back pain and leg pain, ODI, and imaging measurement indicators (spinal canal area, intervertebral bone graft area, intervertebral space height, and degree of intervertebral fusion according to modified Brantigan score).@*RESULTS@#Compared with the Endo-TLIF group, the ULIF group had shorter operation time, but had more intraoperative blood loss and longer hospital stays, with significant differences ( P<0.05). The cerebrospinal fluid leakage occurred in 2 cases of Endo-TLIF group and 1 case of ULIF group, and no other complication occurred. There was no significant difference in the incidence of complications between the two groups ( P>0.05). All patients in the two groups were followed up 12 months. The VAS scores of lower back pain and leg pain and ODI in the two groups significantly improved when compared with those before operation ( P<0.05), and there was no significant difference between different time points after operation ( P>0.05). And there was no significant difference between the two groups at each time point after operation ( P>0.05). Imaging examination showed that there was no significant difference between the two groups in the change of spinal canal area, the change of intervertebral space height, and intervertebral fusion rate at 6 and 12 months ( P>0.05). The intervertebral bone graft area in the ULIF group was significantly larger than that in the Endo-TLIF group ( P<0.05).@*CONCLUSION@#For the patients with lumbar spinal stenosis combined with intervertebral disc herniation, ULIF not only achieves similar effectiveness as Endo-TLIF, but also has advantages such as higher decompression efficiency, flexible surgical instrument operation, more thorough intraoperative intervertebral space management, and shorter operation time.

Studies on the differentiation of human urine derived stem cells into nucleus pulposus-like cells induced by human nucleus pulposus cell exosomes / 中华骨科杂志

Objective:To investigate the effects of exosomes of human nucleus pulposus cells (NPCs) on the differentiation of urine derived stem cells (USCs) into nucleus pulposus-like cells.Methods:USCs and NPCs were isolated and cultured in vitro. The exosomes of NPCs were extracted and detected by Western-blot. USCs cytoplasm was transfected with GFP lentivirus, while nucleus was transfected with DAPI dye. The NPCs exosomes were transfected with PKH26 dye. After co-incubation for 12 h, USCs and NPCs exosomes were observed by macroscopy. USCs differentiation was induced by NPCs exosomes and non-contact co-culture methods. The relative expression of marker gene mRNA of nucleus pulposus cells in each group and the absorbance at 450 nm wavelength were detected.Results:The isolated USCs had the ability to differentiate into osteocytes, adipocytes and chondrocytes with high expression of marker CD29 (99.57%), CD44 (97.46%) and CD73 (97.71%) and with low expression of negative proteins CD31 (0.59%) and CD45 (0.19%). The isolated NPCs highly expressed nuclear pulposus cell marker COL2A1, ACAN and SOX-9. The exosomes extracted from NPCs showed high expression of exosome marker CD63, CD81 and Tsg101. After 12 h co-incubation, NPCs exosomes fused with USCs membrane and appeared in the cytoplasm of USCs. At 3, 5 and 7 days of co-culture, the absorbance value of USCs cells in exosome group (0.44±0.004, 0.76±0.004, 0.82±0.006) was higher than that in co-culture group (0.39±0.022, 0.63±0.035, 0.69±0.012) ( P<0.05). The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (1.80±0.31, 3.50±0.21, 5.35±0.31, 7.46±0.12), COL2A1 (1.43±0.15, 4.33±0.23, 6.89±0.22, 8.11±0.31), SOX-9 (2.21±0.13, 3.13±0.11, 3.96± 0.14, 4.52±0.26) and HIF-1α (1.45±0.16, 2.14±0.21, 4.31±0.41, 4.01±0.25) in exosomes group were significantly higher than those in the control group ( P<0.05) at the 3rd, 7th, 14th and 21st days. The mRNA relative expression of USCs nucleus pulposus marker genes ACAN (5.69±0.21, 6.69±0.13), COL2A1 (6.33±0.17, 7.89±0.15), SOX-9 (4.19±0.29, 4.38±0.12), HIF-1α (4.49±0.32, 4.96±0.26) in exosomes group were significantly higher than those ACAN (3.69±0.35, 5.13±0.23), COL2A1 (3.40±0.16, 6.79±0.19), SOX-9 (2.26±0.32, 3.69±0.26), HIF-1α (2.39±0.11, 3.96±0.13) in non-contact co-culture group ( P<0.05) at the 14th and 21st days. Conclusion:Human nucleus pulposus exosomes could induce differentiation of human USCs into nucleus pulposus-like cells in vitro. Compared with non-contact co-culture, exosomes have higher induction efficiency and can better maintain the proliferation activity of nucleus pulposus-like cells

Correlation between changes of cervical longus and cervical extensor muscles and clinical efficacy after anterior cervical discectomy and fusion / 中华骨科杂志

Objective:To evaluate the volume changes of cervical longus and cervical extensor after anterior cervical discectomy and fusion (ACDF), and the correlation with the clinical efficacy of patients.Methods:All of 57 patients with cervical spondylotic myelopathy who underwent single-segment ACDF surgery from January 2013 to December 2018 were analyzed. The follow-up time was 23.0±4.8 months (range 16-34 months). All included subjects underwent MR examination within 1 week before operation and 3rd, 12th months after operation and at the last follow-up. The axial section cross section area (AxCSA) of the cervical longus and the ratio of length to short diameter line (RLS) at the level of each disc of C 2-C 7 were measured on the axial T2WI. Calculate the volume of the cervical longus based on the layer thickness. At the same time, measure the cervical extensor cross-sectional area (CESA) of the same level including the multifidus, cervical semispinous muscle, semispinous head, splinter head, and cervical splinter muscles, and compare CESA with the corresponding vertebral cross-sectional area (VBA). The ratio is analyzed as the volume of the neck extensor muscle, namely CESA/VBA. At the 3rd and 12th months after operation and at the last follow-up, the axial pain was assessed by visual analogue scale (VAS) for assessing pain, and the modified Japanese Orthopedic Association score (mJOA) and the neck dysfunction index (NDI) were used to assess the functional status of the cervical spine. Analyze the morphological changes of thecervical longus and extensor cervical muscles before and after the operation and during the follow-up period, and analyze the correlation with VAS, mJOA, and NDI. Results:Compared with the preoperative period, the average AxCSA of the surgical segment decreased at the 3rd and 12th months after the operation and at the last follow-up. The difference was statistically significant ( F=24.113, P<0.05), which was changed from 140.84±19.51 mm 2 respectively reduce to 117.74±17.15 mm 2 ( t=6.714, P<0.05), 116.37±18.67 mm 2 ( t=6.841, P<0.05) and 116.27±18.65 mm 2 ( t=6.873, P<0.05). Compared with preoperatively, they were reduced by 16.40%, 17.37% and 17.45%, respectively, while the average RLS of surgical segments increased slightly, and the difference was statistically significant ( F=22.612, P<0.05), which increased from preoperative 1.97±0.67 to 2.73±0.60 (38.58% increased, t=6.380, P<0.05), 2.82±0.64 (43.15% increased, t=6.926, P<0.05) and 2.74±0.62 (39.09% increased, t=6.368, P<0.05). The volume of thecervical longus of the patients decreased after the operation, and the difference was statistically significant ( F=64.511, P<0.05), which decreased from 8853.48±458.65 mm 3 before the operation to 7834.53±461.59 mm 3 (11.51% decreased, t=11.822, P<0.05), 7926.42±456.24 mm 3 (10.47% decreased, t=10.819, P<0.05), 7892.38±450.78 mm 3 (10.86% decreased, t=11.283, P<0.05). There were no statistically significant differences in the non-surgical segment AxCSA, RLS and the volume of thecervical longus at the 3rd and 12th months after surgery and the last follow-up ( P>0.05). There was no statistically significant difference of CESA and CESA/VBA compared to preoperative in the surgical segment and non-surgical segment ( P>0.05). Pearson correlation analysis showed that the volume of cervical longus and VAS at the 3rd month ( r=-0.308, P<0.05), the 12th month ( r=-0.210, P<0.05) and the last follow-up ( r=-0.404, P<0.05) were negatively correlated; Among the volume of cervical longus and NDI in the 3rd month ( r=-0.511, P<0.05), 12th month ( r=-0.518, P<0.05) and the last follow-up ( r=-0.352, P<0.05), there was a negative correlation; However, there was no statistically significant correlation between the cervical longus muscle volume and mJOA at each follow-up time point ( P>0.05); There was no significant correlation between CESA/VBA and VAS, NDI, and mJOA at the 3rd, 12th and last follow-up ( P>0.05). Conclusion:The volume and morphology of cervical longus after ACDF was significantly reduced compared with that before the operation, but the volume and morphology of the cervical extensor muscle did not change significantly. ACDF surgery mainly affects the cervical longus corresponding to the surgical segment, and the volume is negatively correlated with the VAS and NDI during follow-up.

Advances in the treatment of intervertebral disc degeneration by stem cell exosomes / 中华骨科杂志

Exosomes are vesicles with a double globular membrane of lipids that can be secreted by a variety of cells, including stem cells. Exosomes have unique biological characteristics and irreplaceable powerful functions which play an important role in intercellular communication. The various cytokines, signal proteins, lipids and regulatory nucleic acids contained in stem cell exosomes can play a protective role against the injury of kidney, liver, heart, blood vessels and nerves. Stem cell exosomes delay the process of intervertebral disc degeneration by inhibiting the apoptosis of nucleus pulposus cells and increasing the synthesis of extracellular matrix, etc. The mechanism of its role is mainly through miRNA and related signaling pathways. Exosomes contain complex components. Although the mechanism of action of exosomes in intervertebral discs has been preliminarily explored, the components contained in exosomes are complex and the specific situation has not been fully understood, which still needs further study. In this review, the characteristics and functions of stem cell exosomes, extraction, identification and storage methods, the impacttovarious other tissues, as well as the effects on intervertebral discs and their mechanisms were elaborated in order to provide a basis for the study of intervertebral disc degenerative diseases.

Application of DNA barcoding technology to national drug sampling inspection / 药学学报

Adulterants and counterfeits were found in some of the commercial traditional Chinese medicine (TCM) decoctions in Hongjin Xiaojie Jiaonang, Hongjin Xiaojie Pian, and Chaihuang Keli during the national drug sampling inspection. However, it was difficult to determine the species of the adulterants and counterfeits by conventional testing methods. Therefore, a total of 184 samples of the TCM decoctions and raw materials belong to the prescriptions of above mentioned traditional Chinese patent medicines, including Bupleuri Radix, Bajiaolian, Heimayi, and Shufuchong, were collected and authenticated by DNA barcoding technology. 111 ITS2 sequences were obtained from 115 commercial TCM decoctions and raw materials of Bupleuri Radix, among which 71 were Bupleurum chinense, three were B. scorzonerifolium, and 31 were closely related species in the same genus. In addition, counterfeits derived from different genera, such as Ailanthus altissima (one sample), Saposhnikovia divaricate (two samples), and Solidago decurrens (three samples), were also detected. 21 ITS2 sequences were obtained from 22 commercial TCM raw materials of Bajiaolian, among which 15 were Diphylleia sinensis and six were Dysosma versipellis and other species in genus Dysosma. For 22 Heimayi samples, PCR amplification of COI sequence was failed due to genomic DNA degradation. Among 38 Shufuchong samples, 24 COI sequences were obtained and only nine of them were the genuine species (Armadillidium vulgare) recorded in the Chinese Pharmacopoeia, 11 were Porcellio laevis, two were Mongoloniscus sinensis, and two samples could not be identified due to the limitation of database. This study demonstrates that DNA barcoding technology is suitable for the species authentication of the decoctions of traditional Chinese patent medicine prescription. It is a conductive way for the establishment of traceability system for the whole TCM industrial chain.

Management strategy and indications for revisionary internal fixation after percutaneous kyphoplasty/ percutaneous vertebroplasty in cancellous vertebral fractures / 中华创伤骨科杂志

Objective To explore the management strategy and indications for revisionary internal fixation after percutaneous kyphoplasty/percutaneous vertebroplasty (PKP/PVP) in cancellous vertebral fractures.Methods A retrospective analysis was made of the 676 cases of single-segment PKP/PVP at Department of Orthopaedics,The Affiliated Hospital to Qingdao University from January 2008 to January 2019.They were subjected to 4 different managements after their primary PKP/PVP:rehabilitation without any treatment in 637 cases,conservative treatment in 19 cases (including 3 ones who refused any revision),KP/VP revision in 12 cases and internal fixation revision in 8 cases.The rate of volume reduction after bone cement dispersion (Vx) was calculated using software Mimics 17.0 on the basis of primary CT data of all the patients.The correlation regression analysis was made between the revision rate and the approximate quantization value of Vx.The Glasgow Coma Score (GCS) of conscious state was used to evaluate the 39 patients after failure of their primary surgery before the surgical strategy for revision was worked out.The cobb angle,pelvic incidence angle (PI),pelvic inclination angle (PT),sacral inclination angle (SS),sagittal deviation (SVA),pain visual analogue scale (VAS) were measured and recorded before operation and at the last follow-up for the KP/VP revision group and internal fixation revision group,indicated as △cobb,△PI,△PT,△SS,△SVA and △VAS,respectively.The indexes were compared between the 2 groups.Results The incidence of osteoporotic vertebral fractures treated with internal fixation revision was 1.18％ (8/676).The correlation between Vx and revision rate was y =0.53 + 0.04x (P ＜ 0.05).The regression analysis showed that Vx was positively correlated with the revision rate (r2 =0.860,P =0.001) and the fitting curve was correlated (r2 =0.916,P =0.001).The GSC scores revealed 31 normal,6 mild disturbance and 2 moderate disturbance cases.There were no significant differences in gender,age or VAS scores between the KP/VP revision group and the internal fixation revision group (P ＞ 0.05).There was a significant difference in △cobb between the 2 revision groups (6.3° ± 7.5° versus 19.2° ± 14.8°) (P ＜0.05),but there were no significant differences between the 2 groups in △PI (4.1°±5.2° versus 3.3°±6.7°),△PT (0.7°±4.6° versus 0.4° ± 3.2°),△SS (3.7° ± 6.2° versus 3.1° ± 5.3°) or △SVA (-3.2 ± 11.9 mm versus-7.9 ± 9.5 mm) (P ＞ 0.05).Conclusions The outcomes of primary PKP/PVP have a great impact on the decision-making of internal fixation revision.The mode and extent of diffusion after initial vertebral cement perfusion are particularly related to the revision rate.The revision plan should depend on clinical symptoms.The internal fixation revision should be individualized to ensure the quality of life of the patients in line with the principles of "resolving symptoms" and "moderate correction".

Transcriptomic analysis reveals key lncRNAs associated with ribosomal biogenesis and epidermis differentiation in head and neck squamous cell carcinoma / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

OBJECTIVE@#In this study, we aimed to expand current knowledge of head and neck squamous cell carcinoma (HNSCC)-associated long noncoding RNAs (lncRNAs), and to discover potential lncRNA prognostic biomarkers for HNSCC based on next-generation RNA-seq.@*METHODS@#RNA-seq data of 546 samples from patients with HNSCC were downloaded from The Cancer Genome Atlas (TCGA), including 43 paired samples of tumor tissue and adjacent normal tissue. An integrated analysis incorporating differential expression, weighted gene co-expression networks, functional enrichment, clinical parameters, and survival analysis was conducted to discover HNSCC-associated lncRNAs. The function of CYTOR was verified by cell-based experiments. To further identify lncRNAs with prognostic significance, a multivariate Cox proportional hazard regression analysis was performed. The identified lncRNAs were validated with an independent cohort using clinical feature relevance analysis and multivariate Cox regression analysis.@*RESULTS@#We identified nine HNSCC-relevant lncRNAs likely to play pivotal roles in HNSCC onset and development. By functional enrichment analysis, we revealed that CYTOR might participate in the multistep pathological processes of cancer, such as ribosome biogenesis and maintenance of genomic stability. CYTOR was identified to be positively correlated with lymph node metastasis, and significantly negatively correlated with overall survival (OS) and disease free survival (DFS) of HNSCC patients. Moreover, CYTOR inhibited cell apoptosis following treatment with the chemotherapeutic drug diamminedichloroplatinum (DDP). HCG22, the most dramatically down-regulated lncRNA in tumor tissue, may function in epidermis differentiation. It was also significantly associated with several clinical features of patients with HNSCC, and positively correlated with patient survival. CYTOR and HCG22 maintained their prognostic values independent of several clinical features in multivariate Cox hazards analysis. Notably, validation either based on an independent HNSCC cohort or by laboratory experiments confirmed these findings.@*CONCLUSIONS@#Our transcriptomic analysis suggested that dysregulation of these HNSCC-associated lncRNAs might be involved in HNSCC oncogenesis and progression. Moreover, CYTOR and HCG22 were confirmed as two independent prognostic factors for HNSCC patient survival, providing new insights into the roles of these lncRNAs in HNSCC as well as clinical applications.

Long non-coding RNA H19 facilitates bone marrow mesenchymal stem cell survival and vascularization in hypoxic-ischemic conditions in vitro / 中国组织工程研究

BACKGROUND: Our previous study demonstrated that bone marrow mesenchymal stem cells (MSCs) presented with a low survival rate and newly formed vascular-like structures was sparsely distributed in the local infarct tissues after cell transplantation, which certainly impaired the therapeutic efficacy. Long non-coding RNA-H19 (lncRNA-H19) has been confirmed to be associated with MSCs differentiation and mediate vascularization. OBJECTIVE:To observe the influence of lncRNA-H19 on the survival and vascularization potential of MSCs in vitro and to explore the possible mechanism. METHODS:MSCs were obtained and cultured in vitro.Cells were divided into five groups:MSCs+H19,MSCs+H19 negative control (MSCs+H19 NC), MSCs+si-H19, MSCs+si-H19 negative control (MSCs+si-H19 NC) and MSCs groups. MSCs+H19 and MSCs+H19 NC groups were transfected with lncRNA-H19 and lncRNA-H19 scramble RNA respectively, while MSCs+siH19 and MSCs+si-H19 NC groups were transfected with lncRNA-H19 siRNA and lncRNA-H19 siRNA scramble respectively. Cells were cultured under hypoxic-ischemic condition (serum-free medium, 1% O2) for 24 hours. Then, cell proliferation and apoptosis were evaluated using MTS and TUNEL, respectively. Cell supernatant from each experimental group was further co-cultured with human umbilical vein endothelial cells to induce vascularization. The expression of vascular endothelial growth factor A (VEGFA) was thereafter detected using western blot assay. RESULTS AND CONCLUSION: Compared with MSCs+H19 NC and MSCs groups, MSCs+H19 group presented with significantly higher proliferation rate, lower apoptosis percentage and a larger number of vascular branches on matrigel (P < 0.01). There was a significantly higher expression of VEGFA in the MSCs+H19 group than MSCs+H19 NC and MSCs groups. Compared with the MSCs and MSCs+si-H19 NC groups, MSCs+H19 group presented with significantly lower proliferation rate, higher apoptosis percentage and a less number of vascular branches on matrigel (P < 0.01). In addition, VEGFA expression was distinctly downregulated in the MSCs+si-H19 group in comparison with the MSCs+si-H19 NC and MSCs groups. These findings indicate that lncRNA-H19 effectively promotes MSCs survival and vascularization under hypoxic-ischemic condition in vitro,and this effect may be associated with the upregulation of VEGFA.

Simultaneous determination of sixteen mycotoxins contaminants in cogon rootstalk by QuEChERS-UPLC-QqQ mass spectrometry / 中国中药杂志

A method for the simultaneous determination of sixteen mycotoxins in cogon rootstalk was developed using ultra-performance liquid chromatography coupled with triple quadropole mass spectrometry(UPLC-QqQ-MS/MS). The samples were extracted with acetonitrile contained 1% acetic acid and purified by QuEChERS method. The separation was performed on an Agilent Eclipse Plus C₁₈column by gradient elution using methanol and 0.01% aqueous formic acid as mobile phase. The targeted compounds were detected in MRM mode by mass spectrometry with electrospray ionization(ESI)source operated in positive ionization mode. The linear relationships of the sixteen mycotoxins were good in their respective linear ranges. The correlation coefficients(r)ranged among 0.996 2-1.000. The LOQs of the sixteen mycotoxins were between 0.03 and 186.68 μg•kg ⁻¹. The average recoveries ranged from 60.28% to 129.2% with relative standard deviations(RSDs)within 0.29%-11%. The results demonstrated that the proposed method was sensitive and accurate, and suitable for the mycotoxins quantification in cogon rootstalk.

LncRNA-Braveheart promotes the differentiation of bone marrow mesenchymal stem cells in vitro into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND:Our previous work demonstrated that bone marrow mesenchymal stem cells (BMSCs) transplantation could improve cardiac function in rats with myocardial infarction.However,the overall efficacy was unsatisfactory,and there was a low efficiency of BMSCs differentiating into cardiomyocytes in the local infarct myocardium.OBJECTIVE:To transfect long non-coding RNA-Braveheart (IncRNA-Bvht) into BMSCs in order to observe whether it could promote cardiomyocyte differentiation of BMSCs in vitro.METHODS:pLVX-IRES-ZsGreen1-IncRNA-Bvht vector was constructed and applied to transfect IncRNA-Bvht into bMSCs,and then,the transfection efficiency was detected.BMSCs were obtained from C57BL/6 mice and cultured ir vitro.Passage 3 cells were divided into three groups:BMSCs group,null vector group and IncRNA-Bvht group.All cells in the three groups were cultured in the normal condition for 48 hours and cardiomyocytes differentiation was induced by 5-azacytidine for another 24 hours followed by 2-week culture under normal conditions.Cardiomyocyte differentiation of BMSCs was observed under fluorescence microscopy and expression of cardiac specific cell markers including troponin T and myosin were examined using immunofluorescent staining,western blot assay,and qRT-PCR.RESULTS AND CONCLUSION:Cell morphological changes could be observed in all the groups 2 weeks after the induction.Interconnected cells arranged consistently in all the three groups.Immunofiuorescent staining results showed that the expression of troponin T and myosin was notably positive,and the proportion of troponin T positive cells was significantly increased.qRT-PCR and western blot assay results indicated that there were significantly increased levels of troponin T and myosin in the IncRNA-Bvht group as compared with the BMSCs and null vector groups (P ＜ 0.01),suggesting that IncRNA-Bvht could efficiently promote cardiomyocyte differentiation of BMSCs in vitro.

Autotoxicity in Pogostemon cablin and their allelochemicals

Abstract The effects of allelochemicals and aqueous extracts from different Pogostemon cablin (Blanco) Benth., Lamiaceae, parts and rhizosphere soil on growth parameters, leaf membrane peroxidation and leaf antioxidant enzymes were investigated in patchouli. P. cablin seedlings were incubated in solutions containing allelochemicals and aqueous extracts from different patchouli parts and its rhizosphere soil at several concentrations. Firstly, the growth parameters were significantly reduced by the highest concentration of leaves, roots and stems extracts (p < 0.05). As compared to the control, plant height was reduced by 99.8% in the treatment with leaves extracts (1:10). The malondialdehyde content increased greatly when patchouli seedlings were subject to different concentrations of leaves, roots and stems extracts; meanwhile, the superoxide dismutase and peroxidase activities showed an increase trend at the low concentration, followed by a decline phase at the high concentration of roots and leaves extracts (1:10). What's more, leaves and roots extracts had a more negative effect on patchouli growth than stems extracts at the same concentrations. Secondly, the total fresh mass, root length and plant height were greatly reduced by the highest strength of soil extracts. Their decrements were 22.7, 74.9, and 33.1%, respectively. Thirdly, growth parameters and enzymatic activities varied considerably with the kinds of allelochemicals and with the different concentrations. Plant height, root length and total fresh weight of patchouli were greatly reduced by p-hydroxybenzoic acid (200 μM), and their decrements were 77.0, 42.0 and 70.0%, respectively. Finally, three useful measures on reducing the autotoxicity during the sustainable patchouli production were proposed.

Determination of 7 flavonol glycosides in Ginkgo biloba reference extract / 中国中药杂志

Six flavonol glycosides were isolated and calibrated from Ginkgo biloba extract, and then used to calibrate the content in 2 baiches of G. biloba reference extract, so was rutin. RSD values of rutin, kaempferol-3-O-rutinoside, kaempferol-3-O-rhamnoside-2-glu- coside, quercetin-3-O-rhamnop-yranosyl-2-O-(6-O-p-coumaroyl)-glucoside, kaempferol-3-O-rhamnopyranosyl-2-O-(6-O-p-coum-aroyl) - glucoside were around 1.1%-4.6%, nevertheless, RSD values of quercetin-3-O-glucoside and isorhamnetin-3-O-rutinoside were more than 5%. According to the results, the reference extract of G. biloba can be used as the substitute to determine rutin, kaempferol-3-O- rutinoside, kaempferol-3-O-rhamnoside-2-glucoside, quercetin-3-O-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside and kaempferol-3-0-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside instead of corresponding reference substances. So reference extract in place of single component reference in assay is feasible.

Dynamic accumulation of sesquiterpenes in essential oil of Pogostemon cablin

Sesquiterpenes Essential oil produced by patchouli was one of the most important naturally occurring base materials used in the perfume industry, containing various sesquiterpenes. Three different parts (leaves, stems and roots) of Pogostemon cablin (Blanco) Benth., Lamiaceae, were profiled in relation to different maturation phases in this paper, evaluating the variations in content of the major sesquiterpenes in the essential oil. Twelve sesquiterpenes were analyzed by GC-MS throughout the maturity of P. cablin. Patchouli alcohol (37.54%-51.02% in leaves, 28.24%-41.96% in stems and 14.55%-35.12% in roots) was the major sesquiterpene during the maturation of the plant. The average content of several other sesquiterpenes (α-bulnesene, α-guaiene, seychellene, β-humulene and caryophyllene) were higher than 3% among leaves, stems and roots. The content of essential oil, patchouli alcohol, α-bulnesene and several other compounds were highly accumulated at 210 days of maturation after cultivation of P. cablin. Thus, this period was the best moment to exploit the maximum level of these high value-added compounds in P. cablin. Furthermore, our results indicated that the essential oil extracted from leaves of P. cablin has the highest potential to be used in the perfume industry.

Metabolic pathway and metabolites of total diterpene acid isolated from Pseudolarix kaempferi / 药学学报

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

Studies on neuronal tracing with pseudorabies virus / 病毒学报

With its abilities of trans-synaptic tracing and self-replication and wide host range, pseudorabies virus (PRV) has been applied in the field of neuroanatomy since the 1970s. Four decades of PRV application have made many advances in researches on neuronal tracing with PRV. Mechanism studies focused on investigating infection of primary neurons and tracing direction in secondary neurons, while application studies focused on development of new pathological strains and innovation of tracing techniques. To date, the mechanism and application of viral tracing are not completely figured out yet. Integration of molecular biology technology will improve the efficiency in related researches.

Simultaneous determination of eleven flavonoid glycosides in ginkgo biloba leaves collected in different seasons by UPLC PDA method / 药学学报

A new UPLC method was developed for the simultaneous determination of eleven characteristic flavonoid glycosides in Ginkgo biloba leaves. The natural occurrence of flavonoid glycosides in Ginkgo biloba leaves within one vegetative season was investigated for the first time. The analysis was performed on an Agilent ZORBAX Eclipse Plus C18 column (50 mm x 4.6 mm, 1.8 microm), the mobile phase A was acetonitrile, the mobile phase B was 0.4% phosphate aqueous solution in a gradient elution at a flow rate of 0.6 mL x min(-1), the detection was carried out at 360 nm. The result showed that eleven flavonoid glycosides had good linearity with good average recovery, separately. The method was proved to be accurate, rapid and good reproducible for the quality evaluation of Ginkgo biloba leaves, and provide an easy and rapid means for the quantitative analysis of flavonoid glycosides and their content fluctuation with seasons.

Early posterior spinal canal decompression and circumferential reconstruction of rotationally unstable thoracolumbar burst fractures with neurological deficit / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Among the various treatments of neurologically involved unstable thoracolumbar burst fractures, the combination of anterior and posterior instrumentation provides the most stable reconstruction. However, the use of both approaches on a trauma patient may increase the morbidity. This study is a retrospective matched cohort study to evaluate the advantages of a single stage posterior approach for spinal canal decompression in combination with circumferential reconstruction by comparing the clinical and radiographic results.</p><p><b>METHODS</b>From March 2005 to September 2009, patients with matched type spinal fracture, ages at surgery, and involved levels in our institute underwent either a single stage posterior approach (group one, n = 12) or traditional combined approach (group two, n = 14) for spinal canal decompression and circumferential reconstruction were reviewed. Pre- and post-operative X-ray flms were reviewed and changes in Cobb angle of thoracolumbar spine were documented. Intra-operative, post-operative, and general complications were registered.</p><p><b>RESULTS</b>The mean follow-up was (27.7 ± 9.6) months (range, 14 to 56 months) in group one and (29.2 ± 7.4) months (range, 20 to 60 months) in group two (P > 0.05). The mean operation time was 214 minutes (range, 186 ± 327 minutes) in group one and 284 minutes (range, 219 ± 423 minutes) in group two (P < 0.05). The average volume of intraoperative blood loss was 1856 ml (range, 1250 ± 3480 ml) in group one and 2453 ml (range, 1600 ± 3680 ml) in group two (P < 0.05). There was no statistical difference between the groups one and two in average vertebral body height loss at the injured level and the average Cobb angle in sagittal plane before and immediately after surgery. Postoperatively, there was an epidural hematoma in one patient in group one and two patients in group two. Bony union after stabilization was obtained in all patients, without loosening or breakage of screws. Loss of correction (5°) was seen in 1 patient in group one at the 6th month owing to the subsidence of the Titanium mesh cages into the vertebra. In group two, totally four patients suffered respiratory-related complication, including pneumonia in two, severe atelectasis in one and pleural effusions in one. Importantly, there were no intraoperative or postoperative deaths in any group. All patients with incomplete neurologic deficits improved at least 1 Frankel grade.</p><p><b>CONCLUSION</b>Single-stage posterior vertebra resection in combination with circumferential reconstruction is a new option to manage severe thoracolumbar burst fractures.</p>

Proliferation characteristics of a PK-15 cell-adapted strain of porcine parvovirus / 病毒学报

To study the proliferation characteristics of PPV in differently infected way and the variance of concentrations in different cells. A strain of porcine parvovirus(PPV) was adapted to PK-15 cells, and a Real-time fluorescent quantitative PCR (FQ-PCR) assay was developed based on the specific region of the NS1 gene of PPV to quantify the PPV. The FQ-PCR was used to measure the viral concentration of virus-infected cells by simultaneous or step by step inoculation and plot one-step growth curves. The proliferation characteristics of PPV strain in different cells lines (HeLa, MDBK, PK-15 ,ST, F81, BHK-21 and Marc-145) was also compared. The results showed the PK-15 cell -adapted strain of PPV produced CPE after 12 passages, and maintained stable CPE at the following 10 messages. The one-step growth curve showed that the virus concentration of simultaneous inoculation was higher than that of the step-by-step inoculation, and the proliferation cycle of step-by-step inoculation was shorter. The proliferation ability of PPV strain in different cells showed that CPE appeared first inPK-15, followed by ST, HeLa and MDBK, and the virus concentration was highest in ST, followed byPK-15, MDBK and HeLa. NO proliferation was observed in F81, BHK-21 and Marc-145 cells. These findings lay a material foundation for the basic researches on PPV and the development of vaccine.

A review of porcine torovirus research: etiology and epidemiology / 病毒学报

Porcine Torovirus (PToV) is widely distributed in the world with high prevalence rate in swinery. Due to the high detection rate in diarrhea pigs, PToV is thought to be a potential pathogen of swine diarrhea. In recent years, epidemic outbreaks of diarrhea with high morbidity and mortality in China have caused great economic losses. Intertypic recombination events and antigenic cross-reactivity among toroviruses implies potential zoonotic transmission of PToV. The review represented the development history of PToV and made a brief summary of the features in genome and protein epidemiology and laboratory diagnosis of the PToV, and so on.