Stress Writing Textured Graphite Conducting Wires/Patterns in Insulating Amorphous Carbon Matrix as Interconnects.

This study reports a mechanical stress-based technique that involves scratching or imprinting to write textured graphite conducting wires/patterns in an insulating amorphous carbon matrix for potential use as interconnects in future carbonaceous circuits. With low-energy post-annealing below the temperature that is required for the thermal graphitization of amorphous carbon, the amorphous carbon phase only in the mechanically stressed regions transforms into a well aligned crystalline graphite structure with a low electrical resistivity of 420 µΩ-cm, while the surrounding amorphous carbon matrix remains insulating. Micro-Raman spectra with obvious graphitic peaks and high-resolution transmission electron microscopic observations of clear graphitic lattice verified the localized phase transformation of amorphous carbon into textured graphite exactly in the stressed regions. The stress-induced reconstruction of carbon bonds to generate oriented graphitic nuclei is believed to assist in the pseudo-self-formation of textured graphite during low-temperature post annealing.

[Effects of epigallocatechin-3-gallate on the proliferation of colonic cancer cell line SW620 and PAK1 gene expression].

OBJECTIVE: To investigate the effects of epigallocatechin-3-gallate (EGCG) on the proliferation of SW620 cells and the expression of PAK1 gene. METHODS: Human colonic cancer cell line SW620 was treated with EGCG at 40, 60 and 80 micromol/L and cultured in RPMI 1640 medium for 0, 24, 48 and 72 h. The proliferation of SW620 cells was observed by MTT assay before and after EGCG treatment, and the expression of PAK1 protein was observed by Western blotting. RESULTS: SW620 cells treated with EGCG displayed a slowed growth in comparison with the control cells, and the growth rate decreased with the increase of EGCG concentration. PAK1 protein expression was lowered in SW620 cells after EGCG treatment for 48 h. CONCLUSION: EGCG can inhibit the proliferation and partially reduce the expression of PAK1 protein in SW620 cells.

[Effect of p21-activated kinase-1 on the invasiveness of colorectal carcinoma cells in vitro].

OBJECTIVE: To observe the effect of p21-activated kinase-1 (PAK1) gene transfection on the invasiveness of human colorectal carcinoma SW480 cells in vitro. METHODS: SW480 cells in routine culture were transfected with the recombinant plasmid EGFP-C1/PAK1 via Lipofectamine(TM) 2000. The expression of PAK1 protein in SW480 cells was detected using Western blotting, and the changes of the invasiveness of SW480 cells were evaluated using Boyden chamber invasion assay. RESULTS: Forty-eight hours after transfection with pEGFP-C1/ PAK1, the PAK1 protein expression increased significantly in comparison with those in negative and vector control groups. The invasiveness of the SW480 cells was significantly enhanced after the transfection. CONCLUSION: The PAK1 gene transfection can increase the expression of PAK1 in SW480 cells and enhance the invasiveness of the cells. PAK1 can be associated with the invasiveness and metastasis of colorectal carcinoma cells.

[Construction and identification of recombinant Lactococcus lactis highly expressing human granulocyte-macrophage colony stimulating factor].

OBJECTIVE: To transfer human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene into Actococcus lactis and obtain recombinant Lactococcus lactis highly expressing hGM-CSF (LL-CSF). METHODS: The optimized hGM-CSF gene sequence capable of expression in Lactococcus lactis was cloned into the vector pNBC1000, which contained P59 promoter, RBS, MCS, USP45 signal peptide and USP45 stop codon, to generate the recombinant plasmid pNCSF. pNCSF was subcloned into a shuttle vector pTR1001c to acquire the plasmid pTRCSF, which was transferred into Lactococcus lactis to obtain LL-CSF by means of electroporation. SDS-PAGE was used to verify the expression of hGM-CSF protein by the constructed LL-CSF. RESULTS: DNA sequencing and restriction enzyme digestion indicated the successful construction of the recombinant plasmid pNCSF, pTRCSF and the recombinant bacterium LL-CSF that was capable of steady and efficient expression of hGM-CSF as shown by SDS-PAGE. CONCLUSION: The recombinant Lactococcus lactis LL-CSF has been successfully constructed, which can be valuable for studying the biological activity of recombinant hGM-CSF and for evaluating the potential clinical application of the protein.

[PCR-based assembly of the DNA sequence coding for tetanus toxin C fragment].

OBJECTIVE: To develop a PCR-based method for gene assembly of tetanus toxin C fragment (TETC) DNA sequence from a large number of oligodeoxyribonucleotides (oligos). METHODS: To allow for its cloning and expression in Lactococcus lactis, the TETC gene sequence was designed according to the known TETC gene sequence (GenBank accession number M12739, 367-1719) and the amino acid coding in Lactococcus lactis. The sequence contained 1383 nucleotides (nt) with Sal I site added to its 5' end and Xho I and Hind III sites to its 3' end. There were 209 synonymous codon substitutions in the designed gene sequence as compared with the sequence reported in GenBank for amino acid coding in Lactococcus lactis and elimination of the restriction site of EcoR I and Kpn I. The 1380 nt of the sequence was divided into 68 oligos designated as TETC 1 to TETC 68, each containing 40 nt. A 16 nt oligos designated as TETC 69 was designed as the downstream primer. The TETC 1-24 fragment was acquired using the oligos TETC 1 to TETC 24 by PCR-based gene assembly method, and the TETC 23-46 and TETC45-68 fragments were assembled similarly. The full-length TETC gene was assembled using TETC 1 and TETC 69 as the primers when the 3 fragments were mixed. The target gene was gel-purified and digested with Sal I and Hind III, followed by ligation to the pBluescript II SK(+) and digestion with the same enzymes. The positive clones were confirmed by restriction enzyme excision and sequencing. RESULTS: Three 500-bp fragments were acquired by PCR-based gene assembly, and the full-length TETC gene was obtained from the 3 fragment mixed at a equal concentration by a second PCR-based gene assembly using TETC 1 and TETC 69 as the primers. The target gene was cloned to pBluescript II SK(+) vector, and sequence analysis of the positive clones indicated that the assembled sequence was identical to the designed coding sequence of TETC gene. CONCLUSION: PCR-based assembly of the synthesized constitutive gene fragments into the complete sequence can be an effective strategy for synthesis of long DNA sequences in vitro.

[Short hairpin RNA-mediated survivin gene silencing inhibits invasion and metastasis of human colon carcinoma cell line SW480 in vitro].

OBJECTIVE: To investigate the effect of short hairpin RNA (shRNA) targeting survivin on adhesion and invasion of human colon carcinoma cell line SW480 in vitro. METHODS: According to the sequence of the coding region of survivin gene, two strings of 19 nucleotides of inverted sequence flanking the loop sequence of two complementary 9-base oligonucleotides were designed and synthesized to prepare the hairpin construct as the DNA templates for the target shRNA. The shRNA templates were cloned into shRNA expression vector pRNAT-U6.1/Neo, and the resulted vector pRNAT-U6.1/Neo-survivin was transfected into SW480 cells using Lipofectamine 2000. Western blotting was performed to evaluate survivin gene silencing induced by shRNA transfection at the protein level, and the biological behaviors of the SW480 cells were investigated by cell-matrix adhesion, invasion and gelatin-zymography assays. RESULTS: Western blotting revealed significantly lowered survivin protein expression in transfected SW480 cells, and survivin gene silencing induced by shRNA significantly suppressed the metastatic potential of SW480 cells in association with suppressed MMPs activity. CONCLUSIONS: Survivin may play an important role in modulating human colorectal carcinoma cell invasion and metastasis, and survivin gene silencing can inhibit human colorectal cancer cell invasion and the production of MMP-2 and MMP-9. Survivin may affect invasion and metastasis of human colorectal carcinoma cells via regulating the production of MMPs.

[Screening and preliminary analysis of colorectal carcinoma-associated antigen genes].

OBJECTIVE: To screen and identify the genes coding for colorectal carcinoma-associated antigen and analyze the bioinformation of their cDNA sequences. METHODS: Immunoscreening of the cDNA phage-display library derived from human colorectal carcinoma was performed with autologous or allogeneic serum antibody from patients with colorectal cancer through SEREX approach. After amplification of the positive phage clones, the phage DNA was extracted and purified with Qiagen kit, and the fragment sizes of the cDNA of positive clones were identified by PCR and EcoR I and Hind III restriction endonucleases. The cDNAs of the positive clones were ligated into pUCm-T vector and sequenced. The bioinformation of cDNA sequences were analyzed against GenBank+EMBL+DDBJ+PDB Sequences Database. RESULTS AND CONCLUSION: Eleven positive clones were obtained after immunoscreening, and the sizes of the cDNA fragments were 1100, 1300, 1000, 2000, 1200, 1200, 700, 900, 600, 1200 and 1000 bp, respectively, representing 9 antigen genes, including 7 with homology with the known genes. Among the 11 obtained positive clones, 3 were the same cDNA having homology with interferon-induced transmembrance protein-1 and possessing anti-proliferation effect; another 6 represented different genes, namely human BAC clone RP11-453E17 whose function have not been cleared, human cartilage-hair hypoplasia region gene responsible for cartilage-hair hypoplasia, human chromosome 5 clone CTD-2030B15 with insertion mutation, human gene similar to anti tumor necrosis factor-alpha antibody light-chain Fab fragment associated with tumor growth, mRNA of human beta-2-microglobulin in relation to tumor cell proliferation, and human aldolase A gene promoting tumor cell proliferation. The other two cDNA sequences were not identified for homology with currently known genes in GenBank, and their functions awaits further investigation.

[PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 gene].

OBJECTIVE: To study the PCR amplification, cloning and protein expression of interferon-inducible transmembrane protein-1 (IFITMP-1) gene. METHODS: With the cDNA fragment containing IFITMP-1 gene as template, IFITMP-1 gene was amplified using Pfu enzyme by means of PCR. After EcoRI and HindIII digestion, the target gene fragment was linked to pUCm-T plasmid and sequenced. The IFITMP-1 gene was cloned into pET-Trx protein expression plasmid, and the condition for protein expression was optimized. RESULTS: The length of the PCR product of IFITMP-1 gene-containing cDNA fragment was about 1000 bp. The IFITMP-1 gene was successfully inserted into pUCm-T plasmid with correct sequence and cloning of the IFITMP-1 gene into the pET-Trx protein expression plasmid was achieved. Expression of the fusion protein of pUCm-T plasmid and IFITMP-1 gene was detected after IPTG induction. CONCLUSION: Successful amplification and cloning of the IFITMP-1 gene and its protein expression may facilitate further study of the role of IFITMP-1 gene in colorectal cancer.

[Preparation oral liposome-encapsulated recombinant Helicobacter pylori heat shock protein 60 vaccine for prevention of Hp infection].

OBJECTIVE: To prepare oral liposome-encapsulated recombinant Helicobacter pylori (Hp) heat shock protein 60 (Hsp60) vaccine and investigate its effect against Hp infection in mice. METHODS: The recombinant vector PET-22(+)/Hsp60 was transformed into BL21(DE3) E.coli. The recombinant protein was purified with Ni-NTA agrose resin and the oral liposome-encapsulated vaccine was prepared with phosphatidyl choline and cholesterols using film method, with the size distribution of the folate liposomes measured by transmission electronic microscopy. BALB/c mice were divided into 5 groups and immunized by intragastric administration of PBS, liposome, rHsp60 plus choleratoxin (CT), liposome-encapsulated rHsp60, and liposome-encapsulated rHsp60 plus CT, respectively, given once a week for 4 weeks. All the mice were challenged by Hp for 3 times within two weeks following the last immunization and sacrificed 3 weeks after the last challenge. Hp detection was performed by fast urease test. Semi-quantitative assessment of the bacterial colonization density observation of the inflammation severity and gastric histopathology were carried out. RESULTS: The soluble expression product accounted for 27% of the total bacterial protein. The purity of recombinant fusion protein was about 95% after purification. The mean size of the folate liposomes was 0.7+/-0.4 mum. PBS or liposome alone showed no immune-enhancing effect, and rHsp60 plus CT, liposome-encapsulated rHsp60 and liposome-encapsulated rHsp60 plus CT had the protective rates against Hp infection of 73.3%, 66.7% and 86.7%, respectively. The latter 3 preparations effected significantly reduced Hp infection and alleviated the inflammation in the gastric mucosa of the mice challenged with Hp. CONCLUSION: The oral liposome may serve as a potential adjuvant for Hp vaccine in preventing Hp infection.

[Effects of recombinant Helicobacter pylori catalase on oxidative stress in colonic mucosal epithelial cells].

OBJECTIVE: To investigate the effects of recombinant Helicobacter pylori catalase (rHpCAT)on oxidative stress in rat colonic mucosal epithelial cells. METHODS: Oxidative stress model was established by hydroxyl generated from Fenton reaction in cultured colonic mucosal epithelial cells isolated from normal rats, in the model of which the effects of rHpCAT were observed. The cells were divided into normal control, model, 5-aminosalicylic acid (5-ASA, 0.1 mmol/L), and rHpCAT (1 x 10(5), 1 x 10(6), and 1 x 10(7) U/kg, respectively) groups. At the end of the experiment, the content of lactic dehydrogenase (LDH), malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH-Px), catalase (CAT) and, superoxide dismutase (SOD) were detected in the culture supernatant. RESULTS: The contents of LDH, MDA and MPO were elevated while those of GSH-Px, CAT and SOD reduced in the model group. rHpCAT at different doses reduced the release of LDH, depressed the contents of MDA and MPO, and increased the contents of GSH-Px, SOD and CAT. CONCLUSION: rHpCAT has protective effects against rat colonic mucosal oxidative damage.

Effects of probiotic on intestinal mucosa of patients with ulcerative colitis.

AIM: To investigate the effects of probiotic on intestinal mucosae of patients with ulcerative colitis (UC), and to evaluate the role of probiotic in preventing the relapse of UC. METHODS: Thirty patients received treatment with sulphasalazine (SASP) and glucocorticoid and then were randomly administered bifid triple viable capsule (BIFICO) (1.26 g/d), or an identical placebo (starch) for 8 wk. Fecal samples were collected for stool culture 2 wk before and after the randomized treatments. The patients were evaluated clinically, endoscopically and histologically after 2 mo of treatment or in case of relapse of UC. p65 and IkappaB expressions were determined by Western blot analysis. DNA-binding activity of NF-kappaB in colonic nuclear extracts was detected by electrophoretic mobility shift assay (EMSA). mRNA expressions of cytokines were identified by semi-quantitative assay, reverse transcriptase- polymerase chain reaction (RT-PCR). RESULTS: Three patients (20%) in the BIFICO group had relapses during 2-mo follow-up period, compared with 14 (93.3%) in placebo group (P<0.01). The concentration of fecal lactobacilli, bifidobacteria was significantly increased in BIFICO-treated group only (P<0.01). The expressions of NF-kappaB p65 and DNA binding activity of NF-kappaB were significantly attenuated in the treatment group than that in control (P<0.05). The mRNA expression of anti-inflammatory cytokines was elevated in comparison with the control group. CONCLUSION: The probiotic could impede the activation of NF-kappaB, decrease the expressions of TNF-alpha and IL-1beta and elevate the expression of IL-10. These results suggest that oral administration of this new probiotic preparation is effective in preventing flare-ups of chronic UC. It may become a prophylactic drug to decrease the relapse of UC.

Correlation between N 1s XPS binding energy and bond distance in metal amido, imido, and nitrido complexes.

Synchrotron-based X-ray photoelectron spectroscopy is used to measure N 1s binding energies of several metal amido, imido, and nitrido complexes of group 5 and 6 metals. The N 1s binding energy decreases as the formal M-N bond order increases. A simple correlation between the contraction of the M-N bond and the lowering of binding energy is also observed. This correlation supports a notion that the bonding character of a linear metal-imido linkage resembles that of a metal-nitrido linkage closely.

[Construction and identification of the cDNA phage expression library for human colorectal cancer antigens].

OBJECTIVE: To construct a cDNA phage expression library for human colorectal carcinoma antigens. METHODS: After the total RNA was extracted from human colorectal cancer tissues, the single-strand and double-strand cDNA were synthesized through reverse transcriptase PCR and long-distance PCR, with the cDNA fragments smaller than 500 bp removed and the remaining cDNA combined with the right and left arms of dephosphorylated lambdaTriplEx2 phage vector. The recombinant phage were then packaged in vitro by MaxPlax Packaging extract, and a small portion of the packaged phage was used to infect E.coli XL1-Blue. Titer measurement was performed so as to determine the capacity of the library. SfiI restriction endonucleases was used to cut the recombined phage DNA in order to identify the size of inserted cDNA. RESULTS: The constructed cDNA phage expression library for human colorectal cancer antigens consisted of 2.39 x 10(6) pfu/ml bacteriophages with a recombination rate of 97.5% and the length of the inserted cDNA fragment ranged from 600 to 4,000 bp with an average of 1,400 bp. CONCLUSION: The cDNA phage expression library of human colorectal cancer antigens is successfully constructed to meet the currently recognized standards, and can be well applicable in screening cDNA-cloned genes of human colorectal cancer-associated antigens by immunoscreening.