A composite polymer of polystyrene coated with poly(4-vinylpyridine) as a sorbent for the extraction of synthetic dyes from foodstuffs.

A poly(St-co-EGDMA)@poly(4-vinylpyridine-co-EGDMA) composite polymer was synthesised by precipitation reversible addition-fragmentation chain transfer (RAFT) polymerization. The polymer was investigated as a sorbent for extraction of synthetic food dyes: ponceau 4R, tartrazine, sunset yellow, brilliant blue and erythrosine from soft drinks. The morphology and composition of the polymer were characterized and confirmed respectively by scanning electron microscopy and Fourier-transform infrared spectroscopy. The pH dependence experiment revealed that the adsorption of food dyes by the polymer was pH dependent and the maximum adsorption was achieved at pH 3. Adsorption between the polymer and the dyes was mainly due to electrostatic interaction. Under the optimized pH conditions, the polymer was saturated with the dye solutions at a concentration of about 200 µg mL-1 and exhibited a maximum adsorption capacity of 9 µg mg-1. The values were higher than those for polyamide, a sorbent used in the standard method. The recovery from the real samples of the three spiked concentrations 10, 50 and 100 µg mL-1 was respectively within the ranges of 83.2-107.2%, 94.5-110.7% and 79.2-111.5%, with a SD within ±4%. The sorbent could be reused more than 10 times with a recovery higher than 80%. The small volume requirement of the sample and sorbent during the sample pre-treatment, indicated that poly(St-co-EGDMA)@poly(4-vinylpyridine-co-EGDMA) was a potential material for food dye extraction in an environment-friendly and economical manner.

[Effect of long chain non-coding RNA H19 on the migration and invasion of oral cancer cells and its molecular mechanism].

OBJECTIVE: To investigate the effect of the long chain non-coding RNA H19 (lncRNA H19) on the invasion and migration of oral cancer cells and its related molecular mechanism. METHODS: The expression levels of lncRNA H19, miR-107, and cyclin-dependent kinase 6 (CDK6) in the immortalized oral epithelial cell line HIOEC and the oral cancer cell line CAL27 were detected by real-time quantitative polymerase chain reaction. CAL27 cells were transfected with siRNA H19, miR-107 mimics, pcDNA H19, or anti-miR-107, and the effects of H19 and miR-107 on the invasion and migration of cells were examined via Transwell assay. The TargetScan database predicted the targeting of H19, miR-107, and CDK6. Double luciferase reporter gene assay was performed to detect interactions among H19, miR-107, and CDK6. Western blot analysis was conducted to examine the effects of H19 and miR-107 on the protein level of the target gene CDK6. RESULTS: Compared with that in HIOEC cells, the expression of H19 was significantly increased in CAL27 cells (P<0.05). After transfection with siRNA H19, the expression of H19 decreased, and the invasion and migration ability of CAL27 cells were inhibited (P<0.05). H19 could bind specifically to the 3'-UTR of miR-107 to modulate the expression of miR-107. Compared with that in HIOEC cells, the expression of miR-107 significantly decreased in CAL27 cells (P<0.05). The expression of miR-107 increased after transfection with siRNA H19, and anti-mir-107 co-transfection could promote the invasion and migration ability of siRNA H19 in CAL27 cells (P<0.05). Compared with that in HIOEC cells, CDK6 expression significantly increased in CAL27 cells (P<0.05), and the expression level of the gene was coregulated by H19 and miR-107 (P<0.05). CONCLUSIONS: lncRNA H19 plays an important role in the development of oral cancer. It can regulate the invasion and migration of oral cancer cells by targeting the miR-107/CDK6 signaling axis.

Constitutive and Chloroplast Targeted Expression of Acetyl-CoA Carboxylase in Oleaginous Microalgae Elevates Fatty Acid Biosynthesis.

Photosynthetic microalgae are of burgeoning interest in the generation of commercial bioproducts. Microalgae accumulate high lipid content under adverse conditions, which in turn compromise their growth and hinder their commercial potential. Hence, it is necessary to engineer microalgae to mitigate elevated lipid accumulation and biomass. In this study, we identified acetyl-CoA carboxylase (ACCase) in oleaginous microalga Phaeodactylum tricornutum (PtACC2) and expressed constitutively in the chloroplast to demonstrate the potential of chloroplast engineering. Molecular characterization of transplastomic microalgae revealed that PtACC2 was integrated, transcribed and expressed successfully, and localized in the chloroplast. Enzymatic activity of ACCase was elevated by 3.3-fold, and the relative neutral lipid content increased substantially by 1.77-fold, and lipid content reached up to 40.8% of dry weight. Accordingly, the number and size of oil bodies markedly increased. Fatty acid profiling showed that the content of monounsaturated fatty acids increased, while polyunsaturated fatty acids decreased. This method provides a valuable genetic engineering toolbox for microalgal bioreactors with industrial significance.

Synthesis of adenosine-imprinted microspheres for the recognition of ADP-ribosylated proteins.

Core-shell structural adenosine-imprinted microspheres were prepared via a two-step procedure. Polystyrene core particles (CP) were firstly prepared via a reversible addition-fragmentation chain transfer (RAFT) polymerization leaving the iniferter on the surface of the cores, then a molecularly imprinted polymer (MIP) shell was synthesized on the surface of the cores by using acrylamide (AAm) as the functional monomer and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. The formation and growth of the MIP layer were seen dependent on the initiator (AIBN), AAm and the polymerization time used within the polymerization. SEM/TEM images showed that the dimensions of the cores and shells were 2µM and 44nm, respectively. The MIP microspheres exhibited a fast rebinding rate within 2h and a maximum adsorption capacity of 177µg per gram for adenosine. The adsorption fitted a Langmuir-Freundlich (LF) isotherm model with a KLF value of 41mL/µg and a qm value of 177µg/g for the MIP microspheres. The values were larger than those for a non-molecularly imprinted polymer (NIP) particles (5mL/µg and 88µg/g) indicating a better adsorption ability towards adenosine. The MIP microspheres showed a good selectivity for adenosine with a higher adsorption (683nmol/g) for adenosine than that (91nmol/g, 24nmol/g and 54nmol/g) for guanosine, cytidine and uridine respectively. Further experiment proved that the adenosine-imprinted polymer microspheres also had a good selectivity for ADP-ribosylated proteins that the MIP could extract the ADP-ribosylated proteins from the cell extract samples.

Construction of novel chloroplast expression vector and development of an efficient transformation system for the diatom Phaeodactylum tricornutum.

Plastids are ideal subcellular hosts for the expression of transgenes and have been successfully used for the production of different biopolymers, therapeutic proteins and industrial enzymes. Phaeodactylum tricornutum is a widely used aquatic feed species. In this study, we focused on developing a high-efficiency plastid expression system for P. tricornutum. In the plastid transformation vector, the site selected for integration was the transcriptionally active intergenic region present between the trnI and trnA genes, located in the IR (inverted repeat) regions of the plastid genome. Initially, a CAT reporter gene (encoding chloramphenicol acetyltransferase) was integrated at this site in the plastid genome. The expression of CAT in the transformed microalgae conferred resistance to the antibiotic chloramphenicol, which enabled growth in the selection media. Overall, the plastid transformation efficiency was found to be approximately one transplastomic colony per 1,000 microalgae cells. Subsequently, a heterologous gene expression cassette for high-level expression of the target gene was created and cloned between the homologous recombination elements. A TA cloning strategy based on the designed XcmI-XcmI sites could conveniently clone the heterologous gene. An eGFP (green fluorescent protein) reporter gene was used to test the expression level in the plastid system. The relatively high-level expression of eGFP without codon optimisation in stably transformed microalgae was determined to account for 0.12 % of the total soluble protein. Thus, this study presents the first and convenient plastid gene expression system for diatoms and represents an interesting tool to study diatom plastids.

A unique fluorescence response of hexaphenylsilole to methyl parathion hydrolase: a new signal generating system for the enzyme label.

Methyl parathion hydrolase (MPH), an enzyme that catalyses the turnover of methyl parathion (MP) to p-nitrophenol (pNP), can be utilized as an enzyme label. In this paper, a unique fluorescence response of 1,1-bis[4-(diethylaminomethyl)phenyl]-2,3,4,5-tetraphenylsilole (A2HPS) to MPH whose gene was obtained from Pseudomonas sp. strain WBC-3 is described. In the absence of MP, A2HPS could only give a small fluorescence response to the enzyme (I/I0 = 1.1). The detection must be performed under low pH conditions, and the influence of BSA and hemoglobin (Hb) was high; upon addition of the enzyme's substrate, 1 × 10-5µg mL-1 or 2.85 × 10-13 M of the MPH could be reported by A2HPS with a higher I/I0 of 1.7. The detection limit was 105 times more sensitive than that given by a spectrophotometric method. In addition, the assay could be performed at a near neutral pH which was more biocompatible, and little influence was observed from BSA and Hb. The light-on response to the MPH was due to the different quenching effect of the MP and pNP on A2HPS and the improvement in the detection selectivity was due to combining the enzyme reaction with the detection. The findings of this work suggested that A2HPS and MP could form a new reporter system for the MPH enzyme label.

Functional characterization of an ACCase subunit from the diatom Phaeodactylum tricornutum expressed in Escherichia coli.

The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.

[Lymphatic targeting study of pingyangmycin-activated carbon nanoparticles treating oral cancer lymph node metastasis].

OBJECTIVE: To investigate the drug distribution in tissues of cervical lymph node metastasis mice model after submucosa adjacent cancer injection of pingyangmycin-activated carbon nanoparticles (PYM-CH-NP) and evaluate the lymph targeting effect of PYM-CH-NP. METHODS: Pingyangmycin (PYM) was radiolabeled with 125I by modified the chloramine T method. Cervical lymph node metastasis mice model was established by buccal submucosa inoculation of a high lymph metastasis cell line U14 cancer cell. 360 mice models burdened with cervical lymph metastasis were randomly divided into 3 groups. PYM group was treated with PYM water solution, PYM-CH-NP group was treated with PYM-CH-NP. Negative control group was injected with activated carbon nanoparticles. PYM-CH-NP and pingyangmycin water solution were injected in pericancer submucosa of the mice respectively. The radioactivity of drug in blood, heart, liver, spleen, lung, kidney and cervical lymph node were measured after 0.5, 1, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168 h administration. The radioactivity of each samples per unit weight were calculated. The selectivity index (SI) and targeting index (TI) of drug were calculated. RESULTS: The radioactivity of drug in cervical lymph node of PYM-CH-NP group was much higher than PYM group in each time point (P < 0.001), whereas the blood, heart, liver, spleen, lung and kidney uptake of pingyangmycin was greatly decreased in PYM-CH-NP group after 4 h administration (P < 0.001). The SI value of PYM group at each time point was less than 1. While the minimum SI and TI value of PYM-CH-NP was 1.793 and 1.562, the maximum value reached to 68.126 and 14.623 after 72 h administration. CONCLUSION: PYM-CH-NP can increase drug dosage in metastasized cervical lymph nodes, and decrease drug dosage of other organs. So better therapeutic outcome and little adverse reaction may be achieved for lymph node metastasis.

[Effect of Matrine on cell cycle and human telomerase reverse transcriptase of human salivary adenoid cystic carcinoma].

OBJECTIVE: To investigate the effect of the traditional Chinese medicine Matrine on cell cycle and human telomerase reverse transcriptase (hTERT) of human ACC-M cell lines. METHODS: Different concentrations of Matrine were used in the medium of ACC-M cells. Change of cell cycle were detected by flow cytometry after ACC-M cell were cultivated with different concentrations Matrine (0.25, 0.50, 0.75, 1.00 mg x mL(-1)). Expression of hTERT was investigated by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescene and flow cytometry quantitative analysis. RESULTS: Matrine caused obviously the GdG1 phase block and inhibited proliferation of ACC-M cells. At same time, this effect was positive correlation to Matrine concentration and treat time. Matrine can inhibit the expression of hTERT mRNA and protein. CONCLUSION: Matrine can obviously inhibit cell cycle and down-regulate expression of hTERT. Inhibition of cell cycle is possible correlation with down-regulation expression of hTERT.

[Cyst of submandibular gland: report of 1 case and review of the literatures].

This paper reported a 16-year-old girl suffering from painless swelling in the right submandibular region which was diagnosed as ranula, but during the surgery, it was seen that the cyst was closely related to the submandibular gland and it was proved by pathological diagnosis as submandibular cyst. Submandibular cyst and ranula should be distinguished clinically, in order to avoid misdiagnosis and mistreatment.

[The application of three-dimensional reconstruction technique of 64-slices spiral CT in locating of embedded teeth in jaws].

OBJECTIVE: To study the allocation of embedded teeth in jaws using the three-dimensional reconstruction technique of 64-slices spiral CT. METHODS: 27 cases were examined by helical scanning of axial view volume scan CT. The exact localization of the embedded teeth in jaws was acquired by using volume rendering (VR), maximum intensity projection (MIP), multiplanar reformation (MPR) and curve plane reconstruction (CPR). RESULTS: The localization, morphous, size, erupted orientation and relationship between surrounding tissues of the 41 embedded teeth in 27 patients were displayed by effectually using the images of VR, MIP, MPR and CPR. The election of orthodontic treatment or surgical intervention was decided by using 64-slices spiral CT. CONCLUSION: The exact data and objective evidence of the treatment plan could be provided by 64-slices spiral CT which will have important clinical application.

[Diagnosis and choice of surgical methods on traumatic temporomandibular joint ankylosis].

PURPOSE: The purpose of this study was to explore the diagnosis and operative methods for traumatic temporomandibular joint ankylosis. METHODS: Fourteen patients (8 females, 6 males; aged from 7 to 23 years, median age 17.5 years) with traumatic TMJ ankylosis of 1 to 15 years' duration, with a maximal mouth opening(MMO) from 0 to 1.0cm(average:0.46cm) preoperatively were included in this study. They were further divided into two types(I and II) based on false articulation medial to the analysis as shown by coronal computed tomography (CT) coronoid scan. 7 TMJs were treated with medial arthroplasty(MA), in which the lateral fusional bone was removed and the medial false articulation was retained for type I, 6 TMJs were treated with natural positional arthroplasty(NPA) in which the bony fusion between the condyle and glenoid fossa was separated and shaved, the disc was retained and repositioned for type II. Follow-up was carried out and the surgical effect was assessed based on clinical and imaging findings. Student's t test was conducted for comparison with SPSS15.0 software package. RESULTS: There was 9 TMJs from 7 patients for type I and 5 TMJs from 5 patients for type II as shown by CT. The mean postoperative MMO was 3.12cm, and the condylar shape was smooth on CT scan. There was significant difference between preoperative and postoperative MMO, P<0.01. There was no significant difference between MA and NPA, P>0.05. CONCLUSIONS: It is concluded that type I is more common in traumatic TMJ ankylosis than type II. The surgical effect of MA and NPA is good, with less injury, and is worthy of wide application.

[Effect and mechanism of matrine on adhesion and invasion of salivary gland adenoid cystic carcinoma cells in vitro].

PURPOSE: To investigate the effect and mechanism of matrine on adhesion and invasion of human adenoid cystic carcinoma ACC-M cells in vitro. METHODS: MTT assay was used to examine the effect of matrine on proliferation of ACC-M cells treated after 1 day to 3 days. Byden chamber assay was performed to detect the effect of matrine on invasion capacity of the cells; Effect on adhesion potential of ACC-M cells was tested by cell matrigel adhesion assay. Mechanism of inhibitory adhesion and invasion was investigated by expression of E-cad protein. The results was analyzed by SPSS11.0 software package. RESULTS: Matrine could inhibit the proliferation of ACC-M cell lines with apparent dose-dependent and time-dependent effect. Matrine significantly inhibited adhesion and invasion capacity of ACC-M cell lines in vitro with concentration increasing. The expression level of E-cad was significantly increased compared with the untreated group in ACC-M cell lines. CONCLUSIONS: Matrine can inhibit the adhesion and invasion capacity of ACC-M cell lines in vitro. Mechanism of the inhibition effect may be related to over expression of E-cad protein.

[Genetic linkage analysis and mutation detection in Chinese families with basal cell nevus syndrome].

OBJECTIVE: To study the molecular genetic etiology of a Chinese pedigree with basal cell nevus syndrome. METHODS: The proband and his affected mother and a unaffected individual in the pedigree were chosen and peripheral blood was collected from them for DNA. Direct sequencing was performed to detect the mutations of PTCH gene. In order to further confirm the results of sequence analysis, all available family members were analyzed with genetic linkage analysis using 3 highly polymorphic microsatellite DNA markers in the region of 9q22.3-q31. RESULTS: No mutations of PTCH gene was detected in the proband's mother, one synonymous mutation was detected in the proband. Linkage analysis showed that the Lod scores of the 3 markers were: D9S283, Z = -2.11 (theta = 0.00); D9S1690, Z = -2.95 (theta = 0.00); D9S1677, Z = -5.94 (theta = 0.00). CONCLUSIONS: In this pedigree, mutation of PTCH gene is not related to the underlying pathogenesis of the syndrome.

Visualizing the dynamic behavior of poliovirus plus-strand RNA in living host cells.

Dynamic analysis of viral nucleic acids in host cells is important for understanding virus-host interaction. By labeling endogenous RNA with molecular beacon, we have realized the direct visualization of viral nucleic acids in living host cells and have studied the dynamic behavior of poliovirus plus-strand RNA. Poliovirus plus-strand RNA was observed to display different distribution patterns in living Vero cells at different post-infection time points. Real-time imaging suggested that the translocation of poliovirus plus-strand RNA is a characteristic rearrangement process requiring intact microtubule network of host cells. Confocal-FRAP measurements showed that 49.4 +/- 3.2% of the poliovirus plus-strand RNA molecules diffused freely (with a D-value of 9.6 +/- 1.6 x 10(-10) cm2/s) within their distribution region, while the remaining (50.5 +/- 2.9%) were almost immobile and moved very slowly only with change of the RNA distribution region. Under the electron microscope, it was found that virus-induced membrane rearrangement is microtubule-associated in poliovirus-infected Vero cells. These results reveal an entrapment and diffusion mechanism for the movement of poliovirus plus-strand RNA in living mammalian cells, and demonstrate that the mechanism is mainly associated with microtubules and virus-induced membrane structures.

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide ligation assay on DNA chips.

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Improvement of homogeneity of analytical biodevices by gene manipulation.

Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with K(m) and K(cat) similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that the homogeneity of the analytical biodevices could be considerably improved by using the proposed method.

Genetic modification of glucose oxidase for improving performance of an amperometric glucose biosensor.

Glucose oxidase (GOD) was genetically modified by adding a poly-lysine chain at the C-terminal with a peptide linker inserted between the enzyme and poly-lysine chain. The poly-lysine chain was added in order to anchor more electron transfer mediator, ferrocenecarboxylic acid, to GOD for the purpose of improving sensitivity and stability of glucose biosensors. The modified GOD had similar K(m) and K(cat) to those of the wild type enzyme. After interacted with the electron transfer mediator, the modified enzyme retained 90.01% of its native activity, while the commercial GOD and the wild type GOD (Aspergillus niger) retained only 22.43 and 22.17%, respectively. Screen-printed electrodes coated with the modified GOD, wild type yeast-derived GOD or the commercial GOD were tested in glucose solution of different concentrations. Experimental results showed that the biosensor based on the modified GOD gave the largest signal among the three. In addition, the linear range of the biosensor prepared by the modified GOD could extend to 45 mM, while they were about 20 mM for the biosensors based on the wild type yeast-derived enzyme and the commercial enzyme.