A phase conversion headspace technique for the determination of anti-anaerobic activity of drug candidate based on the metabolic acidity change in culture medium.

Screening for anti-anaerobic drug candidates is still challenging although the anaerobic bacteria are important sources for human infections, because the method for anti-anaerobic activity testing is not readily available with low-cost and -expertise. We report a novel method for the determination of the anti-anaerobic activity of drug candidates by automated headspace-gas chromatography (HS-GC). Anaerobic bacteria were inoculated in an anaerobic atmosphere or rapidly using sterile syringe in an air-tight manner, and incubated with and without drugs for 48 h. The metabolic acidities of the cultured media were used as an indicator of cell activities and measured as end-products in place by HS-GC after being completely converted to CO2 with sodium bicarbonate. The present method is precise (relative standard deviation is below 5%) and validated by excellent agreements with a reference method on the determinations of the inhibition rates (root-mean-square error = 10%, n = 48) and half maximal inhibitory concentrations (R2 = 0.996, n = 8) of both pure drug compounds and plant extracts. Advantageously, the present method is sensitive in response to cell activity, safe with regard to cross contamination, and suitable for routine screening of diversified drug candidates for anti-anaerobic activity.

L-Lysine protects against sepsis-induced chronic lung injury in male albino rats.

Sepsis is a severe, life-threatening condition primarily caused by the cellular response to infection. Sepsis leads to increased tissue damage and mortality in patients in the intensive care unit. L-Lysine is an essential amino acid required for protein biosynthesis and is abundant in lamb, pork, eggs, red meat, fish oil, cheese, beans, peas, and soy. Male albino rats were divided into sham, control, 10-mg/kg bwt L-lysine, and 20-mg/kg bwt L-lysine groups. At the end of treatment, we determined the levels of oxidative and inflammatory markers, myeloperoxidase (MPO) and catalase activities, total cell count, the wet/dry ratio of lung tissue, and total protein content. Furthermore, the effect of L-lysine on the cellular architecture of lung tissue was evaluated. L-Lysine significantly reduced the magnitude of lipid peroxidation; total protein content; wet/dry ratio of lung tissue; tumor necrosis factor-alpha, interleukin-8, and macrophage inhibitory factor levels; MPO activity; and total cell, neutrophil, and lymphocyte counts, and it increased the reduced glutathione levels and the glutathione peroxidase, superoxide dismutase, and catalase activities. A normal cellular architecture was noted in rats in the sham group, whereas proinflammatory changes, such as edema and neutrophilic infiltration, were detected in rats in the control group. L-lysine significantly ameliorated these proinflammatory changes. Thus, L-lysine has the potential for the treatment of sepsis-induced CLI.

Comparative and chemometric analysis of correlations between the chemical fingerprints and anti-proliferative activities of ganoderic acids from three Ganoderma species.

INTRODUCTION: Investigation on ganoderic acids (GAs) from different cultivars and origins of Ganoderma species, regarding to their composition, contents and bioactivities, will be of great importance for the development and quality control of Ganoderma-based healthcare products or drugs. OBJECTIVE: Comparative and chemometric analysis of different Ganoderma species were conducted to reveal the correlations between their chemical fingerprints and anti-proliferative activities. METHODOLOGY: Six Ganoderma samples with different origins and parts of fruiting body (pileus and stipe) were extracted with chloroform and enriched by a SPE-C18 cartridge. The eluents were used for high-performance liquid chromatography ultraviolet electrospray ionisation tandem mass spectrometry (HPLC-UV/ESI-MS/MS) analysis and cytotoxicity assay with three cancer cell lines (SGC-7901, HT-29 and Hep G2). Chemometric analysis was applied to correlate their chemical compositions and corresponding bioactivities. RESULTS: Sixteen peaks (accounting for 70% of the total peak areas) were identified as GAs, and their contents ranged from 0.368 to 10.8 µg/g in various Ganoderma species. The extracts from three Ganoderma species had significant anti-proliferative activities (inhibitory rates ranged from 70.8% to 80.7%), and extracts from Lurongzhi showed remarkable inhibition to all three cancer cells (inhibitory rates range from 81.6% to 92.1%). Finally, chemometric analysis revealed that 3,7,12-trihydroxy-4,4,14-trimethyl-11,15-dioxochol-8-en-24-oic acid and 12-acetoxy-15-hydroxy-4,4,14-trimethyl-3,7,11-trioxochol-8-en-24-oic acid were the two compounds with most potential anti-proliferative activity for SGC-7901. CONCLUSION: The correlations between chemical fingerprints and anti-proliferative activities of various Ganoderma species give remarkable insight into the true bioactive components of chemical markers for the quality assessment of the Ganoderma resources, and provide a good guidance for the study on the chemical spectrum-bioactivity relationship.

MHBMDAA: Membrane-based DNA array with high resolution and sensitivity for toxic microalgae monitoring.

Harmful algal blooms (HAB) involving toxic microalgae have posed a serious threat to the marine industry and environment in the past several decades. Efficient techniques are required to monitor the marine environment to provide an effective warning of imminent HAB. Sequenced the partial large subunit rDNA (D1-D2) sequences of eight toxic harmful algae that are commonly distributed along the Chinese coast were cloned. Specific padlock probes (PLP) that contain linker regions composed of universal primer binding sites and Zip sequences were designed from the obtained target DNA. Taxonomic probes complementary to the Zip sequences were tailed and spotted onto a nylon membrane to prepare a DNA array. An optimized multiplex hyperbranched rolling circle amplification (MHRCA) was used to produce biotin-labeled amplified products. Heat-denatured MHRCA products were used to hybridize with DNA array, followed by dot coloration. An MHRCA-based membrane DNA array assay (MHBMDAA) for detecting toxic microalgae was developed. The specificity of the MHBMDAA was confirmed by double cross-reactivity tests of PLP and taxonomic probes. The MHBMDAA was competent for detecting the simulated samples with 103 to 10-1 cells mL-1, which is 10-fold more sensitive than a multiplex PCR-based membrane DNA array. The effectiveness of the MHBMDAA was also validated by testing with natural samples from the East China Sea. Results indicated that the MHBMDAA provides a valuable tool for the sensitive and reliable detection of toxic microalgae for early warning and research purposes.

Physiological and molecular responses of Prorocentrum donghaiense to dissolved inorganic phosphorus limitation.

Prorocentrum donghaiense is an important dinoflagellate as it frequently forms harmful algal blooms that cause serious damage to marine ecosystems and fisheries in the coast of East China Sea. Previous studies showed that phosphorus acquisition (especially inorganic phosphorus) was the limiting factor for P. donghaiense growth. However, the responsive mechanism of this microalga under dissolved inorganic phosphorus (DIP) limitation is poorly understood. In this work, the physiological parameters and differentially expressed genes in P. donghaiense response to DIP limitation were comparatively analyzed. DIP-depleted P. donghaiense displayed decreased growth rate, enlarged cell size, decreased cellular phosphorus content, and high AP activities. A forward suppression subtractive hybridization (SSH) library representing differentially upregulated genes in P. donghaiense under DIP-depleted conditions was constructed, and 134 ESTs were finally identified, with a significant identity (E values<1×10-4) to the deposited genes (proteins) in the corresponding databases. Five representative genes, namely, NAD-dependent deacetylase, phosphoglycolate phosphatase, heat shock protein (HSP) 90, rhodopsin, and HSP40 were investigated through real-time quantitative PCR to verify the effectiveness of the established SSH library. Results showed that all the selected genes were differentially expressed and thus indicated that the established SSH library generally represented differentially expressed genes. These genes were classified into 11 categories according to their gene ontology annotations of biological processes. The members involved in functional responses such as cell defense/homeostasis, phosphorus metabolism, and cellular cycles were specially discussed. This study is the first to perform a global analysis of differentially expressed functional genes in P. donghaiense under DIP-depleted condition. It provided new insights into the molecular adaptive mechanisms of dinoflagellate in response to phosphorous limitation and elucidating the formation mechanism of algal blooms.

Comparative study on alkaloids and their anti-proliferative activities from three Zanthoxylum species.

BACKGROUND: Alkaloids have been considered as the most promising bioactive ingredients in plant species from the genus Zanthoxylum. This study reports on the compositions and contents of the Zanthoxylum alkaloids (ZAs) from three Zanthoxylum species, and their potential anti-proliferation activities. METHODS: An HPLC-UV/ESI-MS/MS method was established and employed to analyze the alkaloids in different Zanthoxylum extracts. The common and unique peaks and their relative contents were summarized and compared to evaluate the similarity and dissimilarity of the three Zanthoxylum species. Meanwhile, inhibitory activity tests to four carcinoma cell lines, i.e., stomach tumor cells (SGC-7901), cervical tumor cells (Hela), colon tumor cells (HT-29) and Hepatic tumor cells (Hep G2), were carried out in vitro to evaluate the bioactivities of the ZAs. RESULTS: Seventy peaks were detected in the crude total alkaloid samples, and 58 of them were identified. As a result, 13 common peaks were found in the extracts of all the three Zanthoxylum species, while some unique peaks were also observed in specific species, with 17 peaks in Z. simulans, 15 peaks in Z. ailanthoides and 11 peaks in Z. chalybeum, respectively. The comparison of the composition and relative contents indicated that alkaloids of benzophenanthridine type commonly present in all the three Zanthoxylum species with high relative contents among the others, which are 60.52% in Z. ailanthoides, 30.52% in Z. simulans and 13.84% in Z. chalybeum, respectively. In terms of activity test, Most of the crude alkaloids extracts showed remarkable inhibitory activities against various tumor cells, and the inhibitory rates ranged from 60.71 to 93.63% at a concentration of 200 µg/mL. However, SGC-7901 cells seemed to be more sensitive to the ZAs than the other three cancer cells. CONCLUSION: The alkaloid profiles detected in this work revealed significant differences in both structures and contents among Zanthoxylum species. The inhibitory rates for different cancer cells in this study indicated that the potential anti-cancer activity should be attributed to quaternary alkaloids in these three species, which will provide great guidance for further exploring this traditional medicinal resource as new healthcare products.

Study on Lesion Assessment of Cerebello-Thalamo-Cortical Network in Wilson's Disease with Diffusion Tensor Imaging.

Wilson's disease (WD) is a genetic disorder of copper metabolism with pathological copper accumulation in the brain and any other tissues. This article aimed to assess lesions in cerebello-thalamo-cortical network with an advanced technique of diffusion tensor imaging (DTI) in WD. 35 WD patients and 30 age- and sex-matched healthy volunteers were recruited to accept diffusion-weighted images with 15 gradient vectors and conventional magnetic resonance imaging (MRI). The DTI parameters, including fractional anisotropy (FA) and mean diffusion (MD), were calculated by diffusion kurtosis estimator software. After registration, patient groups with FA mappings and MD mappings and normal groups were compared with 3dttest and receiver-operating characteristic (ROC) curve analysis, corrected with FDR simulations (p = 0.001, α = 0.05, cluster size = 326). We found that the degree of FA increased in the bilateral head of the caudate nucleus (HCN), lenticular nucleus (LN), ventral thalamus, substantia nigra (SN), red nucleus (RN), right dentate nucleus (DN), and decreased in the mediodorsal thalamus and extensive white matter. The value of MD increased in HCN, LN, SN, RN, and extensive white matter. The technique of DTI provides higher sensitivity and specificity than conventional MRI to detect Wilson's disease. Besides, lesions in the basal ganglia, thalamus, and cerebellum might disconnect the basal ganglia-thalamo-cortical circuits or dentato-rubro-thalamic (DRT) track and disrupt cerebello-thalamo-cortical network finally, which may cause clinical extrapyramidal symptoms.

Flavonoids of Lotus (Nelumbo nucifera) Seed Embryos and Their Antioxidant Potential.

Flavonoids from lotus (Nelumbo nucifera) seed embryos were fractionated over a macroporous resin chromatography into 2 main fractions (I and II), and subsequently identified by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS2 ). Sixteen flavonoids were identified in lotus seed embryos, including 8 flavonoid C-glycosides and 8 flavonoid O-glycosides, in which the flavonoid C-glycosides were the main flavonoids. Among them, 2 flavonoid O-glycosides (luteolin 7-O-neohesperidoside and kaempferol 7-O-glucoside) were identified in lotus seed embryos for the 1st time. For further elucidating the effects of flavonoid C-glycosides to the bioactivities of lotus seed embryos, we compared the differences of the flavonoids and their antioxidant activities between leaves and seed embryos of lotus using the same methods. The results showed the antioxidant activity of flavonoids in lotus seed embryos was comparable or higher than that in lotus leaves, whereas the total flavonoid content in seed embryos was lower than lotus leaves which only contained flavonoid O-glycosides. The flavonoid C-glycosides of lotus seed embryos had higher antioxidant properties than the flavonoid O-glycosides presented in lotus leaves. This study suggested that the lotus seed embryos could be promising sources with antioxidant activity and used as dietary supplements for health promotion.

Identification and characterization of a ferritin gene involved in the immune defense response of scallop Chlamys farreri.

Scallop Chlamys farreri is an important aquaculture species in northern China. However, its mass mortality caused by several pathogens can result in great economic loss and negative impacts to the sustainable development of the scallop industry. Thus, improving the overall understanding of immune response mechanisms involved in host-pathogen interactions is necessary. Ferritins are conserved molecules in organisms that are involved in diverse biological processes, such as mediating host-pathogen responses. In this study, we report a novel ferritin gene from C. farreri (denoted as CfFER). The full length of CfFER is 848 bp and contains a 5'-UTR of 113 bp, a 3'-UTR of 219 bp, and a complete open reading frame (ORF) of 516 bp. The ORF encodes a polypeptide of 171 amino acid residues with a molecular weight of approximately 19.95 kDa and an isoelectric point of 5.07. The CfFER protein exhibited typical ferritin structures, namely, a ferroxidase diiron center, a ferrihydrite nucleation center, and an iron-binding response signature. Phylogenetic analysis revealed that CfFER was closely related to other mollusk ferritin proteins. Expression of CfFER in different tissues was analyzed by quantitative real-time PCR, and results showed that CfFER was ubiquitously expressed in all examined tissues. The highest and lowest expression levels of CfFER were measured in the muscle and hemocyte, respectively. The relative mRNA expression of CfFER in response to bacterial (Vibrio anguillarum) and viral (acute viral necrobiotic virus) challenges sharply increased by ca. 5-fold about12 h post-infection (hpi) and then normalized at 48 hpi. Western blot analysis with polyclonal antibodies generated from the recombinant product of CfFER also demonstrated the presence of ferritin protein in hemocytes. These findings strongly suggest that CfFER is involved in the immune response of C. farreri and protection against pathogen challenge.

Comparative Analysis of Amaryllidaceae Alkaloids from Three Lycoris Species.

The major active constituents from Amaryllidaceae family were reported to be Amaryllidaceae alkaloids (AAs), which exhibited a wide spectrum of biological activities, such as anti-tumor, anti-viral, and acetyl-cholinesterase-inhibitory activities. In order to better understand their potential as a source of bioactive AAs and the phytochemical variations among three different species of Lycoris herbs, the HPLC fingerprint profiles of Lycoris aurea (L. aurea), L. radiata, and L. guangxiensis were firstly determined and compared using LC-UV and LC-MS/MS. As a result, 39 peaks were resolved and identified as AAs, of which nine peaks were found in common for all these three species, while the other 30 peaks could be revealed as characteristic AAs for L. aurea, L. radiata and L. guangxiensis, respectively. Thus, these AAs can be used as chemical markers for the identification and quality control of these plant species. To further reveal correlations between chemical components and their pharmaceutical activities of these species at the molecular level, the bioactivities of the total AAs from the three plant species were also tested against HepG2 cells with the inhibitory rate at 78.02%, 84.91% and 66.81% for L. aurea, L. radiata and L. guangxiensis, respectively. This study firstly revealed that the three species under investigation were different not only in the types of AAs, but also in their contents, and both contributed to their pharmacological distinctions. To the best of our knowledge, the current research provides the most detailed phytochemical profiles of AAs in these species, and offers valuable information for future valuation and exploitation of these medicinal plants.

A novel multiple headspace extraction gas chromatographic method for measuring the diffusion coefficient of methanol in water and in olive oil.

A novel method for the determination of the diffusion coefficient (D) of methanol in water and olive oil has been developed. Based on multiple headspace extraction gas chromatography (MHE-GC), the methanol released from the liquid sample of interest in a closed sample vial was determined in a stepwise fashion. A theoretical model was derived to establish the relationship between the diffusion coefficient and the GC signals from MHE-GC measurements. The results showed that the present method has an excellent precision (RSD<1%) in the linear fitting procedure and good accuracy for the diffusion coefficients of methanol in both water and olive oil, when compared with data reported in the literature. The present method is simple and practical and can be a valuable tool for the determination of the diffusion coefficient of volatile analyte(s) into food simulants from food and beverage packaging material, both in research studies and in actual applications.

[Chemical constituents from Elephantopus tomentosus].

OBJECTIVE: To study the chemical constituents of Elephantopus tomentosus. METHOD: The compounds were isolated by repeated HP20 macro porous adsorption resin column combined with Sephadex LH-20, ODS and silica gel chromatographies. The structures were identified on the basis of extensive spectroscopic data analysis and by comparison of their spectral data reported. RESULT: Eighteen compounds were identified as 2-deethoxy-2beta-hydroxyphantomolin (1), 2beta-hydroxy-2-deethoxy-8-O-deacylphantomolin-8-O-tiglinate (2), 2beta-methoxy-2-deethoxyphantomolin (3), 2beta-methoxy-2-deethoxy-8-O-deacylphantomolin-8-O-tiglinate (4), molephantin (5), molephantinin (6), tricin (7), luteolin (8), quercetin (9), 3beta-friedelinol (10), 3beta-hydroxyolean-12-en-28-oic acid (11), 3, 5-di-O-caffeoyl quinic acid (12), 3,4-di-O-caffeoyl quinic acid (13), syringaresinol-4-beta-D-glucopyranoside (14), xylogranatinin (15), byzantionoside B (16), 2'-hydroxycinnamaldehyde (17), and caffeic acid ethyl ester (18). CONCLUSION: Compounds 9, 11, 14-18 were separated from Elephantopus for the first time.

Antagonist effect of triptolide on AKT activation by truncated retinoid X receptor-alpha.

BACKGROUND: Retinoid X receptor-alpha (RXRα) is a key member of the nuclear receptor superfamily. We recently demonstrated that proteolytic cleavage of RXRα resulted in production of a truncated product, tRXRα, which promotes cancer cell survival by activating phosphatidylinositol-3-OH kinase (PI3K)/AKT pathway. However, how the tRXRα-mediated signaling pathway in cancer cells is regulated remains elusive. METHODOLOGY/PRINCIPAL FINDINGS: We screened a natural product library for tRXRα targeting leads and identified that triptolide, an active component isolated from traditional Chinese herb Trypterygium wilfordii Hook F, could modulate tRXRα-mediated cancer cell survival pathway in vitro and in animals. Our results reveal that triptolide strongly induces cancer cell apoptosis dependent on intracellular tRXRα expression levels, demonstrating that tRXRα serves as an important intracellular target of triptolide. We show that triptolide selectively induces tRXRα degradation and inhibits tRXRα-dependent AKT activity without affecting the full-length RXRα. Interestingly, such effects of triptolide are due to its activation of p38. Although triptolide also activates Erk1/2 and MAPK pathways, the effects of triptolide on tRXRα degradation and AKT activity are only reversed by p38 siRNA and p38 inhibitor. In addition, the p38 inhibitor potently inhibits tRXRα interaction with p85α leading to AKT inactivation. Our results demonstrate an interesting novel signaling interplay between p38 and AKT through tRXRα mediation. We finally show that targeting tRXRα by triptolide strongly activates TNFα death signaling and enhances the anticancer activity of other chemotherapies. CONCLUSIONS/SIGNIFICANCE: Our results identify triptolide as a new xenobiotic regulator of the tRXRα-dependent survival pathway and provide new insight into the mechanism by which triptolide acts to induce apoptosis of cancer cells. Triptolide represents one of the most promising therapeutic leads of natural products of traditional Chinese medicine with unfortunate side-effects. Our findings will offer new strategies to develop improved triptolide analogs for cancer therapy.