Preparation of pH sensitive bacteriostatic W/O/W emulsion microcapsules.

This experiment was done to study the zeolite molecular sieve as a drug-binding effector, the non-antibiotic drug potassium diformate uniformly disperse in the internal aqueous phase of the 'egg box' structure formed by pectin-calcium ions. With oil phase as the intermediate phase and Xanthan gum Chitosan as the external water phase, the W/O/W type sustained release bacteriostatic microcapsules with pH response were prepared and characterized by Fourier transform infrared, thermogravimetric, SEM, and TEM. It can be obtained through characterization experiments that the inner water phase, oil phase, and outer water phase were formed by observation, and W/O/W emulsion microcapsules were obtained and the bacteriostasis effect of microcapsules was verified by bacteriostasis experiment. The permeance experiment showed that the molecular sieve was successfully coated in the microsphere. Studying on drug release mechanism and sustaining release performance of composite emulsion microcapsules. In vitro drug release study showed that the encapsulation efficiency and drug loading rate of microcapsules were improved by adding molecular sieve, reaching 12.31% and 61.55%, respectively. At the same time, we observed that the drug release rate slowed down during the simulated intestinal release process, and the drug release kinetics were in line with the first-order kinetic model and Ritger-Peppas model equation. Experiments had proven that the drug-loaded microcapsules exerted a significant bacteriostatic effect on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis, with the highest antibacterial rates of 97.25%, 94.05%, and 95.93%, respectively. Therefore, the composite emulsion microcapsules can be used as a new controlled-release drug delivery system in vivo.

Construction of pH-sensitive sodium alginates/sodium carboxymethyl cellulose/zeolite P composite hydrogel microspheres loaded with potassium diformate.

As a substitute for feed antibiotics, potassium diformate (KDF) can effectively inhibit bacterial overgrowth in the gastrointestinal tract. To avoid the sudden release of KDF in the stomach, this article proposes a new controlled drug delivery system for controlled drug release. In this system, P-type zeolite molecular sieve (Zeolite P) and drug KDF are combined and embedded into the hydrogel microspheres of sodium alginate (ALG) and sodium carboxymethyl cellulose (CMC). In addition, ALG/CMC/Zeolite P composite hydrogel microspheres were prepared with Ca2+ as the crosslinking agent. The structure, composition, morphology, and thermal stability of the hydrogel microspheres were systematically characterized. The effect of the composition ratio of ALG and CMC on the swelling properties of the hydrogel microspheres was also investigated. The results revealed that ALG and CMC form a hydrogen bond and chelate with Ca2+ to form a double crosslinked network structure. Thus, Zeolite P can be effectively encapsulated in the hydrogel microspheres to form a dense three-dimensional network structure. Particularly, Zeolite P helps in improving the thermal stability of microspheres, balance the swelling properties, and control the release of KDF. The drug release results and release kinetics reveal that the ALG/CMC/Zeolite P composite hydrogel has higher drug release in an environment with pH 7.4. The release kinetics follow the Ritger-Peppas model and the first-order kinetic model, which indicates that the composite hydrogel has good specific pH sensitivity. In vitro antibacterial experiments revealed that the composite hydrogel microspheres have broad-spectrum antibacterial activity, and certain inhibitory effects on Escherichia coli, Staphylococcus aureus, and Bacillus subtilis.

Preparation and properties of environmentally benign waterborne polyurethane composites from sodium-alginate-modified nano calcium carbonate.

Well-dispersed inorganic nanoparticles in organic polymers are critical in the preparation of high-performance nanocomposites. This study prepared a series of waterborne polyurethane (WPU)/calcium carbonate nanocomposites using the solution blending method. Next, FT-IR, TG-DTG and XRD tests were carried out to confirm that the biopolymer sodium alginate (SA) was successfully encapsulated on the surface of the calcium carbonate nanoparticles, and that SA achieved satisfactory surface modification of the calcium carbonate nanoparticles. The Zeta and ultraviolet (UV) absorbance test results reveal that SA-modified nano calcium carbonate (MCC) had good dispersion stability in water. The effects of the MCC dosage on the composite mechanical properties, thermal stability, and cross-sectional morphology observed by scanning electron microscopy, and the water resistance of the nanocomposite were investigated. The results reveal that the incorporation of 3wt% of MCC in WPU had stable distribution, which led to a 54% increase in the tensile strength of the nanocomposite, while maintaining excellent elongation at break (2187%) and increasing the maximum decomposition temperature to 419.6 °C. Importantly, the improved water resistance facilitates the application of this environmentally benign composite material in humid environments.

Two-step hydrothermal synthesis and conversion mechanism of zeolite X from stellerite zeolite.

We investigated the conversion mechanism of stellerite zeolite to zeolite X under two-step hydrothermal conditions. To elucidate the conversion mechanism, solid products were separated from the mixtures at different crystallization times and characterized by XRD, FESEM, FT-IR, Raman, solid-state NMR, XRF, and TEM. The results indicate that in this reaction process, the Si, Al, and Na in the gel solid phases were continuously dissolved and transformed into the gel-liquid-phase. When the concentration of each component reached supersaturation in the gel-liquid-phase, Si, Al, and Na were transferred to the surface of the gel-solid-phase, and nucleation and crystallization occurred on the surface. Abundant nuclei were formed during the second hour of the crystallization. As the crystallization time increased, the nuclei rapidly grew into zeolite X crystals, and the relative crystallinity of zeolite X reached a maximum when the crystallization time reached 4 h. These phenomena indicate that the formation mechanism of zeolite X is a liquid-phase conversion mechanism.

Adsorption of As(V) from Aqueous Solution on Chitosan-Modified Diatomite.

A novel chitosan (CS)-modified diatomite (Dt) was prepared by a simple mixture in the mass ratio to remove As(Ⅴ) from aqueous solution in this research. The CS-modified Dt adsorbent was characterized by scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), and X-ray powder diffraction (XRD) analysis. The parameters to influence the adsorption of As(Ⅴ) ion were studied under such conditions as kinetics, adsorption isotherm, and pH effect. The results revealed that adsorption of As(Ⅴ) was initially rapid and the equilibrium time was reached after 40 min. The optimal value of the pH was 5.0 for better adsorption. The equilibrium data were well fitted to the Langmuir isotherm compared to the Freundlich isotherm, and exhibited the highest capacity and removal efficiency of 94.3% under an initial As(Ⅴ) concentration of 5 mg/L. The kinetic data were well described by the pseudo-second-order model. In addition, 0.1 M NaOH has the best desorption efficiency of As(V) adsorbed on CS-modified Dt, and the removal efficiency of As(V) was still higher than 90% when after six adsorption-desorption cycles. These results showed that the CS-modified Dt could be considered as a potential adsorbent for the removal of As(V) in aqueous solution.

Clostridium beihaiense sp. nov., an anaerobic bacterium isolated from activated sludge.

A Gram-positive, strictly anaerobic, rod-shaped bacterium, designated YB-7T, was isolated from activated sludge of an anaerobic baffled reactor pond in Weizhou terminal wastewater treatment plant, Beihai, Guangxi, China. Strain YB-7T grew at pH 5.0-12.0 (optimum, pH 7.0), 20-45 °C (37 °C) and NaCl concentration of 0-5 % w/v (optimum, 5 %). 16S rRNA gene sequence analysis results showed that strain YB-7T belonged to the genus Clostridium and it was most closely related to Clostridium tetanomorphum DSM 4474T (96.9 % similarity). The DNA-DNA relatedness of strain YB-7T to Clostridium tetanomorphum DSM 4474T was 47.4 %. The DNA G+C content of strain YB-7T was determined to be 32.3 mol%, and the predominant cellar fatty acid (>10 %) was C16 : 0. Polar lipids of strain YB-7T included diphosphatidylglycerol, phosphatidylethylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, two unidentified aminophospholipids, two unidentified phospholipids and unidentified lipids. The results of this study supported the conclusion that strain YB-7T should be assigned to a new member of the genus Clostridium, for which the name Clostridium beihaiense sp. nov. is proposed. The type strain is YB-7T (=CICC 24109T=KCTC 15555T).

Brain contusion as the main risk factor of memory or emotional complaints in chronic complicated mild traumatic brain injury.

OBJECTIVE: To investigate the risk factors for memory or emotional complaints in patients with complicated mild traumatic brain injury (mTBI). METHODS: Retrospective analysis of medical records was conducted by physicians in a teaching hospital in Southern Taiwan, and complicated mTBI had been identified by means of computed tomography. Psychological complaints, including problems with memory and emotions, were collected by structured telephone interviews, 10-15 minutes long, and were held with subjects who agreed to participate in our study. Among 327 patients who were injured for more than two years, 190 agreed to join this study (mean age: 41.6 years; male: 60.5%; stably employed: 50.0%). We used demographic data and neurological factors to predict memory or emotional complaints without muscle power or response speed (MEMR) complaints. RESULTS: Only the presence or absence of cerebral contusions predicted memory or emotional complaints without MEMR complaints in different employed status, and the odds ratio was 4.82-13.50 times higher for those with cerebral contusions than for those without. CONCLUSIONS: Cerebral contusions were the primary risk factor for MEMR complaints in chronic complicated mTBI. Early preventive psychological intervention might be necessary for patients with complicated mTBI and cerebral contusions.

[Soluble expression, purification and bioactivity of recombinant human Era protein].

AIM: To prepare a soluble human Era(hEra) protein and to measure its bioactivity. METHODS: Human era cDNA gene from pUC19 plasmid was subcloned into the expression plasmid pMAL-p2x. pMAL-hEra was transducted to E.coli TB1 and the strain was induced by isopropyl beta-D-thiogalactopyranoside (IPTG). RESULTS: The expressed MBP-fused protein existed in a soluble form. The fused protein made up 23.9% of the total cell lysate. It was purified by amylose affinity chromotography and digested with Factor X. Although the fused segment was dissected, the remained hEra protein was unstable in the solution with the passage of time. The activity assay showed that hEra was a GTPase that could bind GTP and hydrolyze GTP to GDP. CONCLUSION: Human Era protein can be expressed in a soluble form and it has been proved to be a kind of G protein by the experiments in vitro. The study is important to further research into the function of human era gene.

[Construction of shRNA expression vector pWH1 and its function in silencing the HIF1 gene].

AIM: To construct the expression vector of small hairpin RNA(shRNA) and to test its efficacy in silencing the Hypoxia-inducible Factor-1 (HIF1) gene. METHODS: The H1 gene promoter was amplified from the genome of the human blood cells by PCR. Then the promoter was cloned into the pEGFP-C1 vector digested with the restriction enzyme. The constructed vector was named pWH1. The primer was designed to target the human HIF1 cDNA gene. The annealed primer fragment was cloned into pWH1. The new constructed plasmid was transfected into the SGC7901 cell line. Then the expression level of HIF1 gene was assayed by RT-PCR and Western blot. RESULTS: The newly constructed plasmid expressed shRNA to target the HIF1 gene.The results of RT-PCR and Western blot showed the expression of HIF1 gene was reduced dramatically in mRNA and at protein level. CONCLUSION: The successful construction of shRNA expression vector (pWH1) provides a tool for further research into the function of a novel gene.

Expression and regulation of the yggG gene of Escherichia coli.

Our previous study indicated that Era, a membrane-associated GTPase essential for the survival of Escherichia coli, binds with the product of the yggG gene. However, the expression, regulation, and function of the yggG gene have not been established. In this study, the transcript of the yggG gene was determined by analysis of the 5'-end of the yggG mRNA using 5'-RACE (5'-rapid amplification of cDNA ends) method. The promoter and transcription regulatory regions of yggG were analyzed through systematic analysis of the transcriptional activity of the fragment containing a 339-bp 5' flanking sequence of yggG mRNA. The results showed that the sequence -39/-1 upstream of the transcriptional start site of the yggG gene contained a core promoter required for the expression of 25-kDa YggG protein, whereas the -106/-40 region was associated with transcriptional upregulation of yggG under heat shock. Immunocytochemistry and subcellular fraction analysis showed that YggG was a membrane-associated protein. Based on these results, we confirmed that the -39/780 region contains the whole set of the promoter and coding sequence of the yggG gene. The expression regulation of the yggG gene under stress conditions and the YggG protein located on cell membrane are consistent with the bioinformatics analysis that YggG, a metallopeptidase, is functional as a heat shock protein that is associated with Era functions.

Over-expression and siRNA of a novel environmental lipopolysaccharide-responding gene on the cell cycle of the human hepatoma-derived cell line HepG2.

Lipopolysaccharide (LPS) is the toxic determinant for Gram-negative bacterium infection. The individual response to LPS was related to its gene background. It is necessary to identify new molecules and signaling transduction pathways about LPS. The present study was undertaken to evaluate the effects of a novel environmental lipopolysaccharide-responding (Elrg) gene on the regulation of proliferation and cell cycle of the hepatoma-derived cell line, HepG2. By means of RT-PCR, the new molecule of Elrg was generated from a human dental pulp cell cDNA library. Expression level of Elrg in HepG2 cells was remarkably upgraded by the irritation of LPS. Localization of Elrg in HepG2 cells was positioned mainly in cytoplasm. HepG2 cells were markedly arrested in the G1 phase by over-expressing Elrg. The percentage of HepG2 cells in G1 phase partly decreased after Elrg-siRNA. In conclusion, Elrg is probably correlative with LPS responding. Elrg is probably a new protein in cytoplasm which plays an important role in regulating cell cycle. The results will deepen our understanding about the potential effects of Elrg on the human hepatoma-derived cell line HepG2.

Up-regulation of yggG promotes the survival of Escherichia coli cells containing Era-1 mutant protein.

Era is a highly conserved GTPase essential for bacterial growth. Using a digoxigenin-labeled Era protein to screen a phage expression library of Escherichia coli genomic DNA, yggG, a gene that encodes a putative zinc metalloprotease was isolated and characterized. The deduced amino acid sequence of YggG showed high degrees of similarity to some reported heat shock proteins. In this study, the direct interaction between Era and YggG was confirmed, and it was found that the yggG gene, encoding a 25 kDa heat shock protein, was up-regulated at the mRNA level in partially defective Era GTPase mutants (era-1) and in E. coli cells overproducing Era-1. The delta yggG strain displayed the same growth rate as wild-type strain under normal growth conditions and after heat shock. Overexpression of Era-1 in the delta yggG strain resulted in a stronger growth-inhibitory effect than that in the wild-type strain, while coexpression of YggG partially restored the bacterial growth rate. The results indicated that YggG expression is significantly increased in response to stress caused by the Era-1 mutant protein in E. coli, thus promoting the growth of E. coli.

[Expression of mouse lipopolysaccharide response protein LRP in E. coli and preparation of rabbit anti-mLRP serum].

AIM: To express mouse lipopolysaccharide response protein (mLRP) and prepare rabbit anti-mLRP serum. METHODS: The predicted mouse lrp cDNA sequence was obtained by splicing homologous ESTs by comparing human lrp cDNA with mouse ESTs. Then the primers were designed. mlrp cDNA from NIH3T3 cells stimulated with lipopolysaccharide (LPS) was amplified by RT-PCR and was cloned into prokaryotic expression vector pTAT to construct recombinant expression vector pTAT-mlrp. The His-TAT-mLRP fusion protein was expressed in E. coli BL21(DE3) and was used to immunize the rabbits to get rabbit anti-mLRP serum. The anti-serum was purified by the acetone precipitation method. The specificity of the rabbit anti-mLRP serum was determined by Western blot. RESULTS: The predicted length of mlrp cDNA was 1905 bp. The encoding region of the cloned mlrp cDNA, 1554 bp, was inserted into pTAT. The His-TAT-mLRP fusion protein was expressed successfully in E. coli. The rabbit anti-mLRP serum was prepared by immunizing the rabbit with mLRP protein. CONCLUSION: The successful expression of mLRP and the preparation of rabbit anti-mLRP serum lays the foundation for further study of the function of mLRP.

[Expression of a candidate human Era binding protein A19 and the preparation of its polyclonal antibody].

AIM: To express a candidate hEra binding protein A19 in Escherichia coli and to prepare anti-A19 antibody. METHODS: A19 gene was amplified by PCR from the plasmid containing A19 gene and was cloned into the expression vector pGEX-4T3 which was then transformed into E.coli. The A19 protein was expressed under IPTG induction. Antiserum was prepared by immunizing rabbits with the expressed A19 protein. The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: The expressed A19 accounted for about 30.2% of total bacterial protein. The titer of the antiserum was about 1:4 000. Western blot analysis indicated that the antiserum had high specificity. CONCLUSION: A19 fusion protein was highly expressed. The specific anti-A19 antiserum was prepared successfully.

[Expression and purification of two kinds of alternative splicing mouse Era].

AIM: To express and purify two alternative splicing mouse Era proteins and detect whether anti-human Era antibody can be used in the study of mouse Era proteins. METHODS: Two fusion protein expression vectors, pMAL-meraW and pMAL-meraS, were constructed, then the MBP-mEra proteins were expressed in E. coli. The target proteins were purified by amylose affinity chromatography. The specificity of rabbit anti-human Era antibody to the proteins was identified by Western blot. RESULTS: The expressed MBP-mEraW and MBP-mEraS proteins constituted approximately 17% and 19% of the total bacterial proteins. The purity of the fused proteins was 67% and 61% respectively after amylose affinity chromatography. Rabbit anti-human Era antibody had high specificity to these two kinds of splicing mouse Era proteins. CONCLUSION: Two fusion mera genes could be expressed in E. coli by using gene recombination technique. The high specificity of rabbit anti-human Era antibody to the two splicing mouse ERA proteins indicates that this antibody can be used to study the function of these two kinds of splicing mouse Era.

[Preparation of anti-Red antisera and its subcellular localization].

AIM: To prepare rabbit anti-Red antisera. METHODS: The bet, exo and gam genes of lambda phage were amplified by PCR from genomic DNA and cloned into the expression vector pDH2, respectively. Red proteins were induced to express at 42 degrees C. The expressed proteins were analyzed by PAGE and thin-layer scanning. The antisera were prepared by immunizing rabbits with the three Red proteins, respectively. The titers and specificities of the antisera were detected by Western blot. RESULTS: Beta, Exo and Gam proteins accounted for about 40.3%, 49.2% and 73.4% of total bacterial protein, respectively. The titers of the antisera were about 1:2,000. Western blot analysis indicated that the three antisera all had good specificities to the corresponding proteins. CONCLUSION: Specific anti-Red antisera are prepared successfully.

[The expression of truncated YggG protein and preparation of its polyclonal antibody].

AIM: To express truncated YggG protein (TYP) in Escherichia coli and to prepare anti-TYPE antibody. METHODS: Truncated yggg gene was amplified by PCR from the plasmid containing full length yggg gene and was cloned into the expression vector pDH2. The expression of TYP was achieved by thermal induction. Antiserum was prepared by immunizing rabbit with TYP,The titer and specificity of polyclonal antibody were detected by Western blot. RESULTS: Thin-layer scan analysis showed that TYP accounted for about 37.1% of total bacterial protein. The antiserum was about 1:2,000 in titier and highly specific. Full-length rggG protein could also be recognized by the antiserum. CONCLUSION: The TYP was highly expressed, and specific anti-TYP antiserum was prepared successfully.

[High density fed-batch culture of Escherichia coli DH5 alpha/pDH-B2m with DO feed-back control of nutrient feeding].

Optimization of cultivation condition of recombinant E. coli DH5 alpha/pDH-B2m and the condition suitable for expression of recombinant mature peptide of human bone morphogenetic protein-2 was carried out in 500 mL shaking flasks and then transferred to NBS Bioflo IV, a 20 L DO feed-back fed-batch culture system, to obtain rhBMP-2. The results indicate that keeping dissolved oxygen at 40% and controlling nutrient feeding rate with DO feed back strategy can obtain theoretically 3.59 g recombinant mature peptide of hBMP-2 per liter of broth, the final cell density OD600 reaches 57(22.8 g dry cell weight/L), and the expression of rhBMP-2 amounts to 30% of the total protein in E. coli.

Cloning and Optimized Expression of Human Angiogenin in E.coli.

Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli. By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure. The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99. After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE. Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro. Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.