Magnetic dispersive micro-solid phase extraction coupled with dispersive liquid-liquid microextraction followed by graphite furnace atomic absorption spectrometry for quantification of Se(IV) and Se(VI) in food samples.

In this work, magnetic dispersive micro-solid phase extraction (MDMSPE) coupled with dispersive liquid-liquid microextraction (DLLME) was developed for Se(IV) and Se(VI) followed by graphite furnace atomic absorption spectrometry. MDMSPE involved the use of magnetic ZnFe2O4 nanotubes for adsorbing Se(VI). The sorbent was isolated from aqueous phase by using an external magnetic field instead of tedious centrifugation or filtration. In the following step, Se(IV) in the upper aqueous phase of MDMSPE was enriched by DLLME. Samples were prepared with artificial gastric juice to avoid the inter-conversion of target species. The main factors affecting the determination of the analytes were studied in detail. the detection limits of this method were 1.0 and 1.3 pg mL-1 for Se(IV) and Se(VI) with relative standard deviations of 4.6% and 5.1% (c = 1.0 ng mL-1, n = 9), respectively. An enrichment factor of 200 was obtained. This method was used for the detection of Se(IV) and Se(VI) in food samples without any pre-oxidation or pre-reduction operation. A certified reference material of milk powder was analysed by this method, and the determined values were in good agreement with the certified values. Recoveries of spike experiments were in the range of 91.0-107%.

Functional Doxorubicin-Loaded Omega-3 Unsaturated Fatty Acids Nanoparticles in Reversing Hepatocellular Carcinoma Multidrug Resistance.

BACKGROUND This study investigated a nanoparticle drug delivery system to reverse multidrug resistance (MDR) and assessed its anticancer efficacy in hepatocellular carcinoma (HCC). MATERIAL AND METHODS Docosahexaenoic acid (DHA) was used as the functional excipient and doxorubicin (DOX) as the chemotherapeutic drug to synthesize DOX nanoparticles (DOX-nano). The human HCC cell line HepG2 was used for experiments. HepG2/DOX, HepG2+DOX, HepG2+DOX-nano, HepG2/DOX+DOX, and HepG2/DOX+DOX-nano groups cells were treated with DOX or DOX-nano (5 µg/mL). Nude mice bearing a HepG2/DOX xenograft were divided into model, DOX, vector-nano, and DOX-nano groups and injected with saline, DOX reagent, vector-nano, and DOX-nano (2 mg/kg), respectively. Next, cytotoxicity, cellular uptake, cell apoptosis and migration, fluorescence imaging, TUNEL assay, and tumor inhibition effects were assessed in vitro and in vivo. Furthermore, expression of MDR-related proteins was also detected using western blotting. RESULTS Fluorescence imaging showed that the DOX uptake in the DOX-nano-treated group was the strongest in the HCC cells or tumors. Cell apoptosis was significantly increased in DOX-nano-treated HepG2/DOX cells and tumors, and cell migration was significantly inhibited in the DOX-nano-treated HepG2/DOX cells compared with the other groups. The tumor inhibitory rate in DOX-nano-injected tumors was also significantly higher than in other groups. The expression of breast cancer resistance protein, B-cell lymphoma 2, lung resistance protein, multidrug resistance protein, and protein kinase C alpha was significantly decreased in DOX-nano-treated HepG2/DOX cells and xenograft tumors. Significantly better antitumor and MDR-reversing effects were also observed in the HepG2+DOX group compared with the HepG2/DOX group. CONCLUSIONS This study revealed the potential efficacy of a DOX-nano drug delivery system for the treatment of HCC, using HepG2/DOX cells and nude mice bearing HepG2/DOX xenografts.

Extract of bulbus of Fritillaria cirrhosa induces spindle multipolarity in human-derived colonic epithelial NCM460 cells through promoting centrosome fragmentation.

Bulbus of Fritillaria cirrhosa D. Don (BFC), an outstanding antitussive and expectorant herbal drug used in China and many other countries, has potential but less understood genotoxicity. Previously, we have reported that aqueous extract of BFC compromised the spindle assembly checkpoint and cytokinesis in NCM460 cells. Here, we found that one remarkable observation in BFC-treated NCM460 cells was multipolar mitosis, a trait classically compromises the fidelity of chromosome segregation. More detailed investigation revealed that BFC-induced spindle multipolarity in metaphases and ana-telophases in a dose- and time-dependent manner, suggesting BFC-induced multipolar spindle conformation was not transient. The frequency of multipolar metaphase correlated well to that of multipolar ana-telophases, indicating that BFC-induced multipolar metaphases often persisted through anaphase. Unexpectedly, BFC blocked the proliferation of binucleated cells, suggesting spindle multipolarity was not downstream of BFC-induced cytokinesis failure. Exposure of BFC to early mitotic cells, rather than S/G2 cells, contributed greatly to spindle multipolarity, indicating BFC might disrupt centrosome integrity rather than induce centrosome overduplication. The immunofluorescence results showed that the centrosomes were severely fragmented by a short-term treatment of BFC and the extent of centrosome fragmentation in early mitotic cells was larger than this in S/G2 cells. Consistently, several genes (e.g. p53, Rb centrin-2, Plk-4, Plk-1 and Aurora-A) involved in regulating centrosome integrity were significantly deregulated by BFC. Together, our results suggest that BFC causes multipolar spindles primarily by inducing centrosome fragmentation. Coupling these results to our previous observations, we recommend the risk/benefit ratio should be considered in the practical use of BFC.

Isolation, Characterization, and Pharmaceutical Applications of an Exopolysaccharide from Aerococcus Uriaeequi.

Many marine bacteria secrete exopolysaccharides (EPSs), which are made up of a substantial component of the macro-molecules surrounding cells. Recently, the wide demand for EPSs for food, cosmetics, pharmaceutical and other applications has led to great interest in them. In this study, an EPS produced by marine bacteria Aerococcus uriaeequi HZ strains (EPS-A) was isolated and purified to examine its structure and biological function. The molecular weight of EPS-A analyzed by high-performance liquid gel filtration chromatography (HPGFC) is found to have a number average of 2.22 × 105 and weight average of 2.84 × 105, respectively. High-performance liquid chromatography (HPLC) and Fourier-transform⁻infrared (FT⁻IR) analysis indicate that EPS-A was a polysaccharide composed of glucose and a little mannose. In addition, the flocculating rate of sewage of EPS-A was 79.90%. The hygroscopicity studies showed that hygroscopicity of EPS-A was higher than chitosan but lower than that of sodium hyaluronate. The moisture retention of EPS-A showed similar retention activity to both chitosan and sodium hyaluronate. EPS-A also can scavenge free radicals including both OH• free radical and O2•- free radical and the activity to O2•- free radical is similar to vitamin C. Safety assessment on mice indicated that the EPS-A is safe for external use and oral administration. EPS-A has great potential for applications in medicine due to its characteristics mentioned above.

Tenuigenin Prevents IL-1ß-induced Inflammation in Human Osteoarthritis Chondrocytes by Suppressing PI3K/AKT/NF-κB Signaling Pathway.

Tenuigenin (TEN), the main active component of Polygala tenuifolia, has been reported to have anti-inflammatory effects. However, the effects of TEN on IL-1ß-stimulated osteoarthritis chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects and mechanism of TEN on IL-1ß-stimulated human osteoarthritis chondrocytes. Human osteoarthritis chondrocytes were pretreated with or without TEN for 1 h and then stimulated with IL-1ß. The production of NO and PGE2 were detected by the Griess reagent and ELISA. The expression of NF-κB and MAPKs (p38, JNK, ERK) were measured by Western blot analysis. The production of MMP-1, MMP3, and MMP13 were measured by ELISA. The results showed that treatment of TEN significantly inhibited IL-1ß-induced NO and PGE2 production. TEN also suppressed IL-1ß-induced MMP-1, MMP3, and MMP13 expression. Furthermore, TEN was found to inhibit IL-1ß-induced NF-κB activation, PI3K, and AKT phosphorylation. In conclusion, these results suggest that TEN inhibits IL-1ß-induced inflammation in human osteoarthritis chondrocytes by inhibiting PI3K/AKT/NF-κB signaling pathway.

Theoretical and experimental investigation of stability and spectra of doped Ag:ZnSe nanocrystals.

In experiment, doped Ag:ZnSe nanocrystals (NCs) had better stability than that of ZnSe nanocrystals under ambient atmospheres in the presence of air and light illumination. However, it is difficult to explain the mechanism of better stability of Ag:ZnSe nanocrystals from the experiment perspective for doped nanocrystals are more unstable than corresponding pure nanocrystals in general. Using B3LYP/LANL2DZ method, we have investigated the geometrical structures, bonding characters, and molecular orbitals (MOs) of hexagonal and tetrahedral Ag doped ZnSe structures in theory. The results showed that the good stability of Ag:ZnSe nanocrystals can be attributed to the stronger binding between Ag and Se. Moreover, we have proved that Ag doped ZnSe nanocrystals synthesized in experiment should be substituting doped but not vacuity doped. Substituting Ag doped ZnSe molecules have the same configuration as that of the ZnSe structure, but vacuity doped Ag:ZnSe have completely different configuration than ZnSe structure due to the big size of Ag atom. In addition, through contrast of MO of ZnSe and Ag doped ZnSe, we have testified that Ag easily formed bonds with Se. The high binding energy and high probability of forming bonds with Se atom make Ag doped ZnSe nanocrystals have better stability than that of ZnSe nanocrystals.

One-pot synthesis of multicolor MnSe:ZnSe nanocrystals for optical coding.

Though the investigation on controlling the fluorescence properties of nanocrystals (NCs) with single emission has been widely reported, few efforts were spent on adjusting the fluorescence properties of NCs with multiple emission peaks. In this work, we successfully synthesized multicolor MnSe:ZnSe NCs with multiple emission peaks and developed a simple and accurate method to realize photoluminescence (PL) spectra (or color) adjustment. The PL of MnSe:ZnSe NCs has two distinct emission peaks, the trap emission of ZnSe at 475nm and Mn(2+)-induced emission at 585nm. By adjusting the nucleation temperature, the emission color of the NCs can be encoded according to the ratio of the emission intensities at 475 and 585nm. With the nucleation temperature rising from 0 to 70°C, the PL ratio between trap emission and Mn(2+)-induced emission can be consecutively changed from (1, 3) to (1, 0.5). In addition, the trap state is deeply inside the NCs rather than on NCs surface so that the trap emission is stable during environment change. Thus, these MnSe:ZnSe NCs hold great promise as novel single-particle coding labels for biomedical imaging.

Calcitriol enhances 5-aminolevulinic acid-induced fluorescence and the effect of photodynamic therapy in human glioma.

BACKGROUND: Glioma recurrence frequently occurs close to the marginal area of the surgical cavity as a result of residual infiltrating glioma cells. Fluorescence-guided surgery with 5-aminolevulinic acid (ALA) for resection of gliomas has been used as an effective therapeutic approach to discriminate malignant tissue from brain tissue and to facilitate patient prognosis. ALA-based photodynamic therapy is an effective adjuvant treatment modality for gliomas. However, insufficient protoporphyrin IX (PpIX) accumulation may limit the applicability of fluorescence-guided resection and photodynamic therapy in the marginal areas of gliomas. METHODS: To be able to understand how to overcome these issues, human glioma cells and normal astrocytes were used as the model system. Glioma cells and astrocytes were preconditioned with calcitriol for 48 hours and then incubated with ALA. Changes in ALA-induced PpIX fluorescence and cell survival after light exposure were assessed. Furthermore, expression of porphyrin synthetic enzymes in pretreated glioma cells was analyzed. RESULTS: Calcitriol can be administered prior to ALA as a non-toxic preconditioning regimen to significantly enhance ALA-induced PpIX levels and fluorescence. This increase in PpIX level was detected preferentially in glioma versus normal cells. Also, calcitriol pretreated glioma cells exhibited increased cell death following ALA-based photodynamic therapy. Furthermore, mechanistic studies documented that expression of the porphyrin synthesis enzymes coproporphyrinogen oxidase was increased by calcitriol at the mRNA level. CONCLUSION: We demonstrated for the first time a simple, non-toxic and highly effective preconditioning regimen to selectively enhance PpIX fluorescence and the response of ALA-PDT in glioma cells. This finding suggests that the combined treatment of glioma cells with calcitriol plus ALA may provide an effective and selective therapeutic modality to enhance ALA-induced PpIX fluorescent quality for improving discrimination of tumor tissue and PDT efficacy.

Impact of Lycium Barbarum Polysaccharide and Danshensu on vascular endothelial growth factor in the process of retinal neovascularization of rabbit.

AIM: To discuss the impact of Lycium Barbarum Polysaccharide (LBP) and Danshensu purified from Traditional Chinese Medicine (TCM) on vascular endothelial growth factor (VEGF) of rabbits with retinal neovascularization. METHODS: Forty rabbits were divided into normal control group, model control group, LBP group and Danshensu group. Animals in the normal control group were fed in the normal oxygen environment. Animals in the other three groups were put into the environment with 70% oxygen for 5 days in order to build the model of oxygen-induced vascular proliferation retinopathy. And then different TCM extract was injected into the abdominal cavities of these annimals. After 7 days, the VEGF content of in the serum of rabbit was measured by double antibody sandwich method. RESULTS: DATA ANALYSIS INDICATED THAT VEGF CONTENT WAS AS FOLLOWS: Danshensu group was lower than model control group (12.92±3.84ng/L vs 19.32±4.15ng/L, P<0.05); LBP group and normal control group were lower than model control group (12.92±3.84ng/L, 9.26±1.61ng/L vs 19.32±4.15ng/L, P<0.01); total blood viscosity, plasma viscosity, cholesterol content, fibrinogen content and triacylglycerol content after peritoneal injection of LBP and Danshensu were obviously lower than before injection. CONCLUSION: TCM extract-LBP and Danshensu can prominently reduce the content of VEGF in the process of vascular proliferative retinopathy of rabbit; can prevent the occurrence of retinal microvascular disease by improving partial oxygen-deficient environment or affecting all kinds of new growth factor.

Possible applications for fascial anatomy and fasciaology in traditional Chinese medicine.

Research using medical imaging instruments such as computed tomography and magnetic resonance imaging has led to the proposal that the fascial network distributed over the human body is the anatomical basis for the acupoints and meridians of traditional Chinese medicine. Therefore, we put forward a new theory of anatomy called fascial anatomy. In fascial anatomy, a human body is divided into two major systems. One is the supporting-storing system of unspecialized connective tissues. The other is a functional system. An undifferentiated non-specific connective tissue network, with the participation of the nervous and the immune systems, constitutes the supporting-storing system of the human body. The various differentiated functional cells in the body that are supported and surrounded by the supporting-storing system constitute the functional system. The discipline that studies the supporting-storing system and the mutual relationship between this system and the functional system in a living human body is called fasciaology. The establishment of fascial anatomy and fasciaology opens a new research field in anatomy; consequently, fasciaology will play a significant role in biological medicine and traditional Chinese medical research, as well as future clinical practice.

Loss of astrocytic glutamate transporters in Wernicke encephalopathy.

Wernicke encephalopathy (WE), a neurological disorder caused by thiamine deficiency (TD), is characterized by structural damage in brain regions that include the thalamus and cerebral cortex. The basis for these lesions is unclear, but may involve a disturbance of glutamatergic neurotransmission. We have therefore investigated levels of the astrocytic glutamate transporters EAAT1 and EAAT2 in order to evaluate their role in the pathophysiology of this disorder. Histological assessment of the frontal cortex revealed a significant loss of neurons in neuropathologically confirmed cases of WE compared with age-matched controls, concomitant with decreases in alpha-internexin and synaptophysin protein content of 67 and 52% by immunoblotting. EAAT2 levels were diminished by 71% in WE, with levels of EAAT1 also reduced by 62%. Loss of both transporter sites was confirmed by immunohistochemical methods. Development of TD in rats caused a profound loss of EAAT1 and EAAT2 in the thalamus accompanied by decreases in other astrocyte-specific proteins. Treatment of TD rats with N-acetylcysteine prevented the downregulation of EAAT2 in the medial thalamus, and ameliorated the loss of several other astrocyte proteins, concomitant with increased neuronal survival. Our results suggest that (1) loss of EAAT1 and EAAT2 glutamate transporters is associated with structural damage to the frontal cortex in patients with WE, (2) oxidative stress plays an important role in this process, and (3) TD has a profound effect on the functional integrity of astrocytes. Based on these findings, we recommend that early treatment using a combination of thiamine AND antioxidant approaches should be an important consideration in cases of WE.

Rapid baseline separation of enantiomers and a mesoform of all-trans-astaxanthin, 13-cis-astaxanthin, adonirubin, and adonixanthin in standards and commercial supplements.

Astaxanthin (3,3'-dihydroxy-beta,beta-carotene-4,4'-dione) is widely used as important colorant in the aquaculture feed industry, and as nutraceuticals in human health products. Synthetic all-trans-astaxanthin consists of a mixture of a pair of enantiomers (3R,3'R and 3S,3'S) and a mesoform (3R,3'S). A high-performance liquid chromatography (HPLC) method for direct, rapid, and baseline separation of three stereoisomers of all-trans-astaxanthin is described for the first time on an immobilized cellulosic column (Chiralpak IC). Enantiomers of two important precursors in the biosynthetic pathway of astaxanthin, adonirubin and adonixanthin, were also directly separated. In addition, the major cis form of astaxanthin (13-cis-astaxanthin) resulted from isomerization was isolated with preparative C18 separation, and the separation of all four stereoisomers of 13-cis-astaxanthin is achieved. Finally, a stereoisomeric purity test of commercial astaxanthin supplements confirmed that they were from a natural source, although their levels were quite low.

[Explanation of essence and substance basis of channels and collaterals with fasciology].

In our research of digital human body, construct of image structures close to the recorder of human channels and collaterals, and trace back to development biology of connective tissue and process of organic evolution, and it is concluded that connective tissue bracket in human body may constitute a new functional system--human self-supervision and control system, meanwhile, we propose a new study field, fasciology. The significance is that it introduces a new functional system in human body and develops new field of scientific research; and it annotate biological foundation and therapeutic mechanisms of traditional Chinese medicine therapy, which provides medical biological foundation for modern channel research of traditional Chinese medicine; it proposes a suggestion for development strategy of traditional Chinese medicine.

[From meridians and acupoints to self-supervision and control system: a hypothesis of the 10th functional system based on anatomical studies of digitized virtual human].

Chinese acupuncture and moxibustion has been widely accepted as a useful therapeutics all over the world, but its mechanism has not been fully defined. For this purpose, a reticular framework of whole-body fascia and connective tissues has been established by means of digitized virtual human technique. The virtual acupoints represented three-dimensionally were compared with the sites for stimulation in practice of traditional Chinese medicine (TCM) acupuncture therapy. The results showed that the fascial network constituted by the connective tissues may be the anatomical basis for acupuncture therapy. We found that the acupoints were mainly located where thick connective tissues were present. In this fascial network, sensitive nerve endings, active cells and lymphatic vessels abounded in the sites with thick connective tissue, and needling at these sites induced definite biological effects. In light of biological phylogeny and embryo development, we believe that the connective tissue network may constitute a new functional system in the human body, the Self-supervision and control system (Fasciology), which provides a theoretical base for acupuncture therapy.

[Study on the meridians and acupoints based on fasciaology: an elicitation of the study on digital human being].

OBJECTIVE: To explore the correlativity of meridians and acupoints with fascial system. METHODS: The lines and beads-like structures seen by fasciaology and scanning connective tissue, were combined with traditional meridians and collaterals, and acupoints to investigate channels, collaterals and acupoints. RESULTS: The high correlativity of the meridians and acupoints with the fascial system was found. CONCLUSION: The concept, functions, clinical application and mechanisms of meridians and acupoints can be preliminarily explained.

Downregulation of complexin I and complexin II in the medial thalamus is blocked by N-acetylcysteine in experimental Wernicke's encephalopathy.

Metabolic dysfunction as a consequence of thiamine (vitamin B1) deficiency (TD), a model of Wernicke's encephalopathy, leads to elevation of extracellular glutamate concentration in vulnerable brain regions consistent with the development of excitotoxicity. Complexin I and complexin II are two genes labeling principally inhibitory and excitatory synapses, respectively. Because current evidence supports an important role for complexins in the modulation of neurotransmitter release, we examined the involvement of both proteins in the pathology of the medial thalamus and inferior colliculus in TD rats by immunoblotting. At the symptomatic stage, complexin I and complexin II levels in the medial thalamus were decreased by 63% and 45%, respectively, compared to control animals, but were unchanged in the inferior colliculus. These changes in thalamus were also observed using immunohistochemical methods, and seemed to be due to downregulation of both proteins because synaptophysin levels were unaffected in this brain region. In addition, cotreatment with the antioxidant N- acetylcysteine prevented both neuronal loss and downregulation of complexins. Our findings suggest dysregulation of excitatory and inhibitory neurotransmitter release in the medial thalamus, which is not present in the inferior colliculus. Furthermore, loss of complexin I and II in the thalamus may be mediated by processes that involve oxidative stress. Such changes in complexin levels may contribute to the pathophysiology of thalamic damage in TD, and offer a potential basis for the well-known differences in pathology between this structure and the inferior colliculus in this disorder.

Effect of five flavonoid compounds isolated from leaves of Diospyros kaki on stimulus-induced superoxide generation and tyrosyl phosphorylation of proteins in human neutrophils.

BACKGROUND: The crude drug "kaki-yô" is a traditional medicine used in Japan as a hypotensive drug. METHODS: The effect of five flavonoid compounds isolated from the leaves of Diospyros kaki was investigated on the stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils. The five compounds examined were kaempferol 3-O-beta-D-galactopyranoside (TR), kaempferol 3-O-beta-D-glucopyranoside (AS), isorhamnetin 3-O-beta-D-glucopyranoside (IS), quercetin 3-O-beta-D-galactopyranoside (HY), quercetin 3-O-beta-D-glucopyranosyl-(6-->1)-alpha-L-rhamnopyranoside (RU). RESULTS: When the cells were preincubated with these five compounds, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed in a concentration-dependent manner. The arachidonic acid (AA)-induced superoxide generation was suppressed by TR, AS, HY and RU. On the other hand, the superoxide generation was weakly enhanced by IS in low concentration (5-20 micromol/l), but was suppressed in high concentration (50 micromol/l). The superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) suppressed the TR, IS, HY and RU, but AS gave no effect. When the cells were incubated with fMLP in the presence of TR, IS and RU, fMLP-induced tyrosyl phosphorylation of 45-kDa proteins of the cells was dose-dependently suppressed in parallel to the suppression of fMLP-induced superoxide generation. These five flavonoids showed almost no hemolytic effect even at a concentration of 500 micromol/l. CONCLUSION: Flavonoid compounds suppressed stimulus-induced superoxide generation and tyrosyl phosphorylation and may have pharmaceutical application.

Effect of five triterpenoid compounds isolated from leaves of Diospyros kaki on stimulus-induced superoxide generation and tyrosyl phosphorylation in human polymorphonuclear leukocytes.

BACKGROUND: The crude drug "kaki-yô" is a traditional medicine used in Japan as a hypotensive drug. METHODS: The effect of five triterpenoid compounds, isolated from leaves of Diospyros kaki on stimulus-induced superoxide generation and phosphorylation of tyrosine residues of protein in human neutrophils was investigated. The five compounds examined were alpha-amyrin (A), uvaol (UV), ursolic acid (UA), 19 alpha-hydroxy ursolic acid (HU) and 19 alpha,24-dihydroxy ursolic acid (DHU). RESULTS: When the cells were preincubated with these compounds, the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) was significantly suppressed in a concentration-dependent manner. These compounds also suppressed the superoxide generation induced by arachidonic acid (AA) in high concentrations. In the case of the superoxide generation induced by phorbol 12-myristate 13-acetate (PMA), UA, HU and DHU suppressed the superoxide generation but A and UV gave no effect. When the cells were incubated with fMLP in UA, HU and DHU, fMLP-induced tyrosyl phosphorylation of 45 kDa proteins of the cells was dose-dependently suppressed in parallel to the suppression of fMLP-induced superoxide generation. CONCLUSIONS: Triterpenoid compounds suppress stimulus-induced superoxide generation and tyrosyl phosphorylation and may have pharmaceutical applications.