Determination of schizandrin in rat plasma by high-performance liquid chromatography-mass spectrometry and its application in rat pharmacokinetic studies

A sensitive liquid chromatography-mass spectrometric (LC/MS) method for the quantification of schizandrin in rat plasma was developed and validated after solid-phase extraction (SPE). Chromatographic separation was achieved on a reversed-phase Shimadzu C(18) column with the mobile phase of acetonitrile-sodium acetate (10 micromol/L) and step gradient elution resulted in a total run time of about 11.7 min. The analytes were detected using an electrospray positive ionization mass spectrometry in the selected ion monitoring (SIM) mode. A good linear relationship was obtained in the concentration range studied (0.005-2.000 microg/mL) (r=0.9999). Lower limit of quantification (LLOQ) was 5 ng/mL and the lower limit of detection (LLOD) was 2 ng/mL using 100 microL plasma sample. Average recoveries ranged from 75.85 to 88.51% in plasma at the concentrations of 0.005, 0.100 and 1.000 microg/mL. Intra- and inter-day relative standard deviations were 5.95-12.93% and 3.87-14.53%, respectively. This method was successfully applied for the pharmacokinetic studies in rats.     

4-Hydroxynonenal modulation of p53 family gene expression in the SK-N-BE neuroblastoma cell line

4-Hydroxynonenal (HNE), a product of lipid peroxidation, inhibits proliferation of several tumor cells. The p53 tumor suppressor protein plays a critical role in cell cycle control, by inducing p21 expression, and in apoptosis, by inducing bax expression. Recently, two other proteins with many p53-like properties, TAp73 (p73) and TAp63 (p63), have been discovered. SK-N-BE human neuroblastoma cells express the three p53 family proteins and can be used for the study of their induction. We investigated HNE action in the control of proliferation, differentiation, and apoptosis in SK-N-BE cells and the HNE effect on the expression of p53, p63, p73, p21, bax, and G1 cyclins. Retinoic acid (RA) was used as a positive control. HNE inhibited cell proliferation without inducing differentiation; it decreased S-phase cells and increased the number of apoptotic cells. RA reduced the proportion of S-phase cells and did not induce apoptosis. HNE increased p53, p73, p63, p21, and bax expression at different time points. HNE reduced cyclin D2 expression and the phosphorylation of pRb protein. Our results demonstrated that HNE inhibits SK-N-BE cell proliferation by increasing the expression of p53 family proteins and p53 target proteins which modulate cell cycle progression and apoptosis.     

A Systematic Comparison of the Anti-Tumoural Activity and Toxicity of the Three Adv-TKs

Adenovirus 5 vectors, known respectively as, the first generation, second generation and oncolytic adenovirus, have been studied extensively in preclinical and clinical trials. However, hitherto few systemic evaluations of the efficacy and toxicity of these adenoviral vectors that have reflected the vertical history of adenovirus based cancer gene therapy strategies have been undertaken. This study has chosen Adv-TK, the well-established adjuvant treatment in cancer, and compared its efficacy and safety with those of the two newly synthesized oncolytic adenovirus vectors encoding the HSV-TK gene, namely M7 and M8. The results obtained showed that systemic administration of 1×10⁸ pfu M7 had an anti-tumour efficacy similar to that of 3×10¹⁰ pfu Adv-TK whilst M8 performed even better. Furthermore, compared to Adv-TK, M7 and M8 reduced the incidence of metastases and substantially prolonged the survival time of the mice xenografted with human orthotopic gastric carcinomas with disseminated metastasis. Even more exciting, however, were the similar toxic and immune safety results obtained from the administration of high doses of M7 or M8 in comparison with Adv-TK in immunocompetent and permissive syrian hamster. The data here exhibit a comprehensive display of the efficacy and safety of the three mutants and provide evidence for the future preclinical use of the M7 and M8 viruses.     

Tyramide signal amplification for antibody-overlay lectin microarray: a strategy to improve the sensitivity of targeted glycan profiling

Antibody-overlay lectin microarray (ALM) has been used for targeted glycan profiling to identify disease-related protein glycoforms. In this context, high sensitivity is desired because it allows for the identification of disease-related glycoforms that are often present at low concentrations. We describe a new tyramide signal amplification (TSA) for the antibody-overlay lectin microarray procedure for sensitive profiling of glycosylation patterns. We demonstrate that TSA increased the sensitivity of the microarray over 100 times for glycan profiling using the model protein prostate specific antigen (PSA). The glycan profile of PSA enriched from LNCAP cells, obtained at a subnanogram level with the aid of TSA, was consistent with the previous reports. We also established the glycan profile of prostate specific membrane antigen (PSMA) using the TSA and ALM. Thus, the TSA for antibody-overlay lectin microarray is a sensitive, rapid, comprehensive, and high-throughput method for targeted glycan profiling and can potentially be used for the identification of disease-related protein glycoforms.     

Critical role of VCP/p97 in the pathogenesis and progression of non-small cell lung carcinoma

Background

Valosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.

Methodology/Principal Findings

Our results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).

Conclusions/Significance

Thus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.     

肠道微生态干预对非酒精性脂肪性肝病患者的影响：系统评价和网状meta分析

评估肠道微生态干预对成年非酒精性脂肪性肝病（NAFLD）患者的肝脏特异性、代谢、炎症和肠道微生物群影响的最新证据。

检索Embase、PubMed、Cochrane Library、Web of Science、中国知网、万方数据库,筛选符合条件的随机临床试验,对纳入研究的数据进行提取和分析。

纳入37项随机对照试验：20项评估益生菌,2项评估益生元,10项评估合生元,2项评估益生菌和益生元,3项评估益生菌、益生元和合生元,治疗时间7～48周。经网状meta分析,对于大部分临床指标,益生元与合生元组干预效果优于对照组,且呈现出时间相关性,但肠道微生态干预各组干预效果差异无统计学意义。

网状meta分析表明肠道微生态干预改善了NAFLD的临床指标和肠道微生态,提示合生元可能是一种优于益生菌和益生元的干预措施,但需要足够的剂量和干预时间,我们的研究建议至少连续使用24周。

     

In Vitro Propagation and Reintroduction of the Endangered Renanthera imschootiana Rolfe

Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l⁻¹ BA, 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ banana homogenate (BH), and 1.0 g l⁻¹ AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.     

MiR-181b-5p Downregulates NOVA1 to Suppress Proliferation,Migration and Invasion and Promote Apoptosis in Astrocytoma

MicroRNAs (miRNAs) are small, short noncoding RNAs that modulate the expression of numerous genes by targeting their mRNA. Numerous abnormal miRNA expression patterns are observed in various human malignancies, and certain miRNAs can act as oncogenes or tumor suppressors. Astrocytoma, the most common neuroepithelial cancer, represents the majority of malignant brain tumors in humans. In our previous studies, we found that the downregulation of miR-181b-5p in astrocytomas is associated with a poor prognosis. The aim of the present study was to investigate the functional role of miR-181b-5p and its possible target genes. miR-181b-5p was significantly downregulated in astrocytoma specimens, and the reduced expression of miR-181b-5p was inversely correlated with the clinical stage. The ectopic expression of miR-181b-5p inhibited proliferation, migration and invasion and induced apoptosis in astrocytoma cancer cells in vitro. The NOVA1 (neuro-oncological ventral antigen 1) gene was further identified as a novel direct target of miR-181b-5p. Specifically, miR-181b-5p bound directly to the 3''-untranslated region (UTR) of NOVA1 and suppressed its expression. In clinical specimens, NOVA1 was overexpressed, and its protein levels were inversely correlated with miR-181b-5p expression. Furthermore, the changing level of NOVA1 was significantly associated with a poor survival outcome. Similar to restoring miR-181b-5p expression, downregulating NOVA1 inhibited cell growth, migration and invasion. Overexpression of NOVA1 reversed the inhibitory effects of miR-181b-5p. Our results indicate that miR-181b-5p is a tumor suppressor in astrocytoma that inhibits tumor progression by targeting NOVA1. These findings suggest that miR-181b-5p may serve as a novel therapeutic target for astrocytoma.