Counter-current chromatography for the separation of terpenoids: A comprehensive review with respect to the solvent systems employed

Copyright @ 2014 The Authors.This article is distributed under the terms of the Creative Commons Attribution License which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.Natural products extracts are commonly highly complex mixtures of active compounds and consequently their purification becomes a particularly challenging task. The development of a purification protocol to extract a single active component from the many hundreds that are often present in the mixture is something that can take months or even years to achieve, thus it is important for the natural product chemist to have, at their disposal, a broad range of diverse purification techniques. Counter-current chromatography (CCC) is one such separation technique utilising two immiscible phases, one as the stationary phase (retained in a spinning coil by centrifugal forces) and the second as the mobile phase. The method benefits from a number of advantages when compared with the more traditional liquid-solid separation methods, such as no irreversible adsorption, total recovery of the injected sample, minimal tailing of peaks, low risk of sample denaturation, the ability to accept particulates, and a low solvent consumption. The selection of an appropriate two-phase solvent system is critical to the running of CCC since this is both the mobile and the stationary phase of the system. However, this is also by far the most time consuming aspect of the technique and the one that most inhibits its general take-up. In recent years, numerous natural product purifications have been published using CCC from almost every country across the globe. Many of these papers are devoted to terpenoids-one of the most diverse groups. Naturally occurring terpenoids provide opportunities to discover new drugs but many of them are available at very low levels in nature and a huge number of them still remain unexplored. The collective knowledge on performing successful CCC separations of terpenoids has been gathered and reviewed by the authors, in order to create a comprehensive document that will be of great assistance in performing future purifications. © 2014 The Author(s)

A simulation study for comparing testing statistics in response-adaptive randomization

<p>Abstract</p> <p>Background</p> <p>Response-adaptive randomizations are able to assign more patients in a comparative clinical trial to the tentatively better treatment. However, due to the adaptation in patient allocation, the samples to be compared are no longer independent. At large sample sizes, many asymptotic properties of test statistics derived for independent sample comparison are still applicable in adaptive randomization provided that the patient allocation ratio converges to an appropriate target asymptotically. However, the small sample properties of commonly used test statistics in response-adaptive randomization are not fully studied.</p> <p>Methods</p> <p>Simulations are systematically conducted to characterize the statistical properties of eight test statistics in six response-adaptive randomization methods at six allocation targets with sample sizes ranging from 20 to 200. Since adaptive randomization is usually not recommended for sample size less than 30, the present paper focuses on the case with a sample of 30 to give general recommendations with regard to test statistics for contingency tables in response-adaptive randomization at small sample sizes.</p> <p>Results</p> <p>Among all asymptotic test statistics, the Cook's correction to chi-square test (<it>T</it>_<it>MC</it>) is the best in attaining the nominal size of hypothesis test. The William's correction to log-likelihood ratio test (<it>T</it>_<it>ML</it>) gives slightly inflated type I error and higher power as compared with <it>T</it>_<it>MC</it>, but it is more robust against the unbalance in patient allocation. <it>T</it>_{<it>MC </it>}and <it>T</it>_{<it>ML </it>}are usually the two test statistics with the highest power in different simulation scenarios. When focusing on <it>T</it>_{<it>MC </it>}and <it>T</it>_<it>ML</it>, the generalized drop-the-loser urn (GDL) and sequential estimation-adjusted urn (SEU) have the best ability to attain the correct size of hypothesis test respectively. Among all sequential methods that can target different allocation ratios, GDL has the lowest variation and the highest overall power at all allocation ratios. The performance of different adaptive randomization methods and test statistics also depends on allocation targets. At the limiting allocation ratio of drop-the-loser (DL) and randomized play-the-winner (RPW) urn, DL outperforms all other methods including GDL. When comparing the power of test statistics in the same randomization method but at different allocation targets, the powers of log-likelihood-ratio, log-relative-risk, log-odds-ratio, Wald-type Z, and chi-square test statistics are maximized at their corresponding optimal allocation ratios for power. Except for the optimal allocation target for log-relative-risk, the other four optimal targets could assign more patients to the worse arm in some simulation scenarios. Another optimal allocation target, <it>R</it>_{<it>RSIHR</it>}, proposed by Rosenberger and Sriram (<it>Journal of Statistical Planning and Inference</it>, 1997) is aimed at minimizing the number of failures at fixed power using Wald-type Z test statistics. Among allocation ratios that always assign more patients to the better treatment, <it>R</it>_{<it>RSIHR </it>}usually has less variation in patient allocation, and the values of variation are consistent across all simulation scenarios. Additionally, the patient allocation at <it>R</it>_{<it>RSIHR </it>}is not too extreme. Therefore, <it>R</it>_{<it>RSIHR </it>}provides a good balance between assigning more patients to the better treatment and maintaining the overall power.</p> <p>Conclusion</p> <p>The Cook's correction to chi-square test and Williams' correction to log-likelihood-ratio test are generally recommended for hypothesis test in response-adaptive randomization, especially when sample sizes are small. The generalized drop-the-loser urn design is the recommended method for its good overall properties. Also recommended is the use of the <it>R</it>_{<it>RSIHR </it>}allocation target.</p

Long-term safety and outcome of a temporary self-expanding metallic stent for achalasia: a prospective study with a 13-year single-center experience

To prospectively evaluate the long-term clinical safety and efficacy of a newly designed self-expanding metallic stent (SEMS) in the treatment of patients with achalasia. Seventy-five patients with achalasia were treated with a temporary SEMS with a 30-mm diameter. The SEMSs were placed under fluoroscopic guidance and removed by gastroscopy 4–5 days after stent placement. Follow-up data focused on dysphagia score, technique and clinical success, clinical remissions and failures, and complications and was performed at 6 months, 1 year, and within 3 to 5 years, 5 to 8 years, 8 to 10 years, and >10 years postoperatively. Stent placement was technically successful in all patients. Complications included stent migration (n = 4, 5.33%), chest pain (n = 28, 38.7%), reflux (n = 15, 20%), and bleeding (n = 9, 12%). No perforation or 30-day mortality occurred. Clinical success was achieved in all patients 1 month after stent removal. The overall remission rates at 6 months, 1, 1–3, 3–5, 5–8, 8–10, and >10 year follow-up periods were 100%, 96%, 93.9%, 90.9%, 100%, 100%, and 83.3%, respectively. Stent treatment failed in six patients, and the overall remission rate in our series was 92%. The median and mean primary patencies were 2.8 ± 0.28 years (95% CI: 2.25–3.35) and 4.28 ± 0.40 years (95% CI: 3.51–5.05), respectively. The use of temporary SEMSs with 30-mm diameter proved to be a safe and effective approach for managing achalasia with a long-term satisfactory clinical remission rate

HDAC Inhibition Decreases the Expression of EGFR in Colorectal Cancer Cells

Epidermal growth factor receptor (EGFR), a receptor tyrosine kinase which promotes cell proliferation and survival, is abnormally overexpressed in numerous tumors of epithelial origin, including colorectal cancer (CRC). EGFR monoclonal antibodies have been shown to increase the median survival and are approved for the treatment of colorectal cancer. Histone deacetylases (HDACs), frequently overexpressed in colorectal cancer and several malignancies, are another attractive targets for cancer therapy. Several inhibitors of HDACs (HDACi) are developed and exhibit powerful antitumor abilities. In this study, human colorectal cancer cells treated with HDACi exhibited reduced EGFR expression, thereby disturbed EGF-induced ERK and Akt phosphorylation. HDACi also decreased the expression of SGLT1, an active glucose transporter found to be stabilized by EGFR, and suppressed the glucose uptake of cancer cells. HDACi suppressed the transcription of EGFR and class I HDACs were proved to be involved in this event. Chromatin immunoprecipitation analysis showed that HDACi caused the dissociation of SP1, HDAC3 and CBP from EGFR promoter. Our data suggested that HDACi could serve as a single agent to block both EGFR and HDAC, and may bring more benefits to the development of CRC therapy

Integrated analysis of DNA methylation and gene expression reveals specific signaling pathways associated with platinum resistance in ovarian cancer

<p>Abstract</p> <p>Background</p> <p>Cisplatin and carboplatin are the primary first-line therapies for the treatment of ovarian cancer. However, resistance to these platinum-based drugs occurs in the large majority of initially responsive tumors, resulting in fully chemoresistant, fatal disease. Although the precise mechanism(s) underlying the development of platinum resistance in late-stage ovarian cancer patients currently remains unknown, CpG-island (CGI) methylation, a phenomenon strongly associated with aberrant gene silencing and ovarian tumorigenesis, may contribute to this devastating condition.</p> <p>Methods</p> <p>To model the onset of drug resistance, and investigate DNA methylation and gene expression alterations associated with platinum resistance, we treated clonally derived, drug-sensitive A2780 epithelial ovarian cancer cells with increasing concentrations of cisplatin. After several cycles of drug selection, the isogenic drug-sensitive and -resistant pairs were subjected to global CGI methylation and mRNA expression microarray analyses. To identify chemoresistance-associated, biological pathways likely impacted by DNA methylation, promoter CGI methylation and mRNA expression profiles were integrated and subjected to pathway enrichment analysis.</p> <p>Results</p> <p>Promoter CGI methylation revealed a positive association (Spearman correlation of 0.99) between the total number of hypermethylated CGIs and GI₅₀values (<it>i.e</it>., increased drug resistance) following successive cisplatin treatment cycles. In accord with that result, chemoresistance was reversible by DNA methylation inhibitors. Pathway enrichment analysis revealed hypermethylation-mediated repression of cell adhesion and tight junction pathways and hypomethylation-mediated activation of the cell growth-promoting pathways PI3K/Akt, TGF-beta, and cell cycle progression, which may contribute to the onset of chemoresistance in ovarian cancer cells.</p> <p>Conclusion</p> <p>Selective epigenetic disruption of distinct biological pathways was observed during development of platinum resistance in ovarian cancer. Integrated analysis of DNA methylation and gene expression may allow for the identification of new therapeutic targets and/or biomarkers prognostic of disease response. Finally, our results suggest that epigenetic therapies may facilitate the prevention or reversal of transcriptional repression responsible for chemoresistance and the restoration of sensitivity to platinum-based chemotherapeutics.</p

The Effects of Sleep Hypoxia on Coagulant Factors and Hepatic Inflammation in Emphysematous Rats

OBJECTIVES: To develop a sleep hypoxia (SH) in emphysema (SHE) rat model and to explore whether SHE results in more severe hepatic inflammation than emphysema alone and whether the inflammation changes levels of coagulant/anticoagulant factors synthesized in the liver. METHODS: Seventy-five rats were put into 5 groups: SH control (SHCtrl), treated with sham smoke exposure (16 weeks) and SH exposure (12.5% O(2), 3 h/d, latter 8 weeks); emphysema control (ECtrl), smoke exposure and sham SH exposure (21% O(2)); short SHE (SHEShort), smoke exposure and short SH exposure (1.5 h/d); mild SHE (SHEMild), smoke exposure and mild SH exposure (15% O(2)); standard SHE (SHEStand), smoke exposure and SH exposure. Therefore, ECtrl, SHEShort, SHEMild and SHEStand group were among emphysematous groups. Arterial blood gas (ABG) data was obtained during preliminary tests. After exposure, hepatic inflammation (interleukin -6 [IL-6] mRNA and protein, tumor necrosis factor α [TNFα] mRNA and protein) and liver coagulant/anticoagulant factors (antithrombin [AT], fibrinogen [FIB] and Factor VIII [F VIII]) were evaluated. SPSS 11.5 software was used for statistical analysis. RESULTS: Characteristics of emphysema were obvious in emphysematous groups and ABGs reached SH criteria on hypoxia exposure. Hepatic inflammation parameters and coagulant factors are the lowest in SHCtrl and the highest in SHEStand while AT is the highest in SHCtrl and the lowest in SHEStand. Inflammatory cytokines of liver correlate well with coagulant factors positively and with AT negatively. CONCLUSIONS: When SH is combined with emphysema, hepatic inflammation and coagulability enhance each other synergistically and produce a more significant liver-derivative inflammatory and prothrombotic status

Functional Identification and Characterization of the Brassica Napus Transcription Factor Gene BnAP2, the Ortholog of Arabidopsis Thaliana APETALA2

BnAP2, an APETALA2 (AP2)-like gene, has been isolated from Brassica napus cultivar Zhongshuang 9. The cDNA of BnAP2, with 1, 299 bp in length, encoded a transcription factor comprising of 432 amino acid residues. Results from complementary experiment indicated that BnAP2 was completely capable of restoring the phenotype of Arabidopsis ap2-11 mutant. Together with the sequence and expression data, the complementation data suggested that BnAP2 encodes the ortholog of AtAP2. To address the transcriptional activation of BnAP2, we performed transactivation assays in yeast. Fusion protein of BnAP2 with GAL4 DNA binding domain strongly activated transcription in yeast, and the transactivating activity of BnAP2 was localized to the N-terminal 100 amino acids. To further study the function of BnAP2 involved in the phenotype of B. napus, we used a transgenic approach that involved targeted RNA interference (RNAi) repression induced by ihp-RNA. Floral various phenotype defectives and reduced female fertility were observed in B. napus BnAP2-RNAi lines. Loss of the function of BnAP2 gene also resulted in delayed sepal abscission and senescence with the ethylene-independent pathway. In the strong BnAP2-RNAi lines, seeds showed defects in shape, structure and development and larger size. Strong BnAP2-RNAi and wild-type seeds initially did not display a significant difference in morphology at 10 DAF, but the development of BnAP2-RNAi seeds was slower than that of wild type at 20 DAF, and further at 30 DAF, wild-type seeds were essentially at their final size, whereas BnAP2-RNAi seeds stopped growing and developing and gradually withered

Nestin-GFP Transgene Reveals Neural Precursor Cells in Adult Skeletal Muscle

Background: Therapy for neural lesions or degenerative diseases relies mainly on finding transplantable active precursor cells. Identifying them in peripheral tissues accessible for biopsy, outside the central nervous system, would circumvent the serious immunological and ethical concerns impeding cell therapy. Methodology/Principal Findings: In this study, we isolated neural progenitor cells in cultured adult skeletal muscle from transgenic mice in which nestin regulatory elements control GFP expression. These cells also expressed the early neural marker Tuj1 and light and heavy neurofilament but not S100b, indicating that they express typical neural but not Schwann cell markers. GFP+/Tuj1+ cells were also negative for the endothelial and pericyte markers CD31 and a-smooth muscle actin, respectively. We established their a) functional response to glutamate in patch-clamp recordings; b) interstitial mesenchymal origin; c) replicative capacity; and d) the environment necessary for their survival after fluorescenceactivated cell sorting. Conclusions/Significance: We propose that the decline in nestin-GFP expression in muscle progenitor cells and its persistence in neural precursor cells in muscle cultures provide an invaluable tool for isolating a population of predifferentiated neural cells with therapeutic potential

Histological and transcriptome-wide level characteristics of fetal myofiber hyperplasia during the second half of gestation in Texel and Ujumqin sheep

<p>Abstract</p> <p>Background</p> <p>Whether myofibers increase with a pulsed-wave mode at particular developmental stages or whether they augment evenly across developmental stages in large mammals is unclear. Additionally, the molecular mechanisms of myostatin in myofiber hyperplasia at the fetal stage in sheep remain unknown. Using the first specialized transcriptome-wide sheep oligo DNA microarray and histological methods, we investigated the gene expression profile and histological characteristics of developing fetal ovine longissimus muscle in Texel sheep (high muscle and low fat), as a myostatin model of natural mutation, and Ujumqin sheep (low muscle and high fat). Fetal skeletal muscles were sampled at 70, 85, 100, 120, and 135 d of gestation.</p> <p>Results</p> <p>Myofiber number increased sharply with a pulsed-wave mode at certain developmental stages but was not augmented evenly across developmental stages in fetal sheep. The surges in myofiber hyperplasia occurred at 85 and 120 d in Texel sheep, whereas a unique proliferative surge appeared at 100 d in Ujumqin sheep. Analysis of the microarray demonstrated that immune and hematological systems' development and function, lipid metabolism, and cell communication were the biological functions that were most differentially expressed between Texel and Ujumqin sheep during muscle development. Pathways associated with myogenesis and the proliferation of myoblasts, such as calcium signaling, chemokine (C-X-C motif) receptor 4 signaling, and vascular endothelial growth factor signaling, were affected significantly at specific fetal stages, which underpinned fetal myofiber hyperplasia and postnatal muscle hypertrophy. Moreover, we identified some differentially expressed genes between the two breeds that could be potential myostatin targets for further investigation.</p> <p>Conclusions</p> <p>Proliferation of myofibers proceeded in a pulsed-wave mode at particular fetal stages in the sheep. The myostatin mutation changed the gene expression pattern in skeletal muscle at a transcriptome-wide level, resulting in variation in myofiber phenotype between Texel and Ujumqin sheep during the second half of gestation. Our findings provide a novel and dynamic description of the effect of myostatin on skeletal muscle development, which contributes to understanding the biology of muscle development in large mammals.</p