Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer.

How mutations or dysfunction of CFTR may increase the risk of malignancies in various tissues remains an open question. Here we report the interaction between CFTR and an adherens junction molecule, AF-6/afadin, and its involvement in the development of colon cancer. We have found that CFTR and AF-6/afadin are co-localized at the cell-cell contacts and physically interact with each other in colon cancer cell lines. Knockdown of CFTR results in reduced epithelial tightness and enhanced malignancies, with increased degradation and reduced stability of AF-6/afadin protein. The enhanced invasive phenotype of CFTR-knockdown cells can be completely reversed by either AF-6/afadin over-expression or ERK inhibitor, indicating the involvement of AF-6/MAPK pathway. More interestingly, the expression levels of CFTR and AF-6/afadin are significantly downregulated in human colon cancer tissues and lower expression of CFTR and/or AF-6/afadin is correlated with poor prognosis of colon cancer patients. The present study has revealed a previously unrecognized interaction between CFTR and AF-6/afadin that is involved in the pathogenesis of colon cancer and indicated the potential of the two as novel markers of metastasis and prognostic predictors for human colon cancer.

A Microtitre Plate Dilution Method for Minimum Killing Concentration Is Developed to Evaluate Metabolites-Enabled Killing of Bacteria by ß-lactam Antibiotics.

Because, as of yet, there are few new antibiotics active against multidrug-resistant bacteria are being explored, compounds including metabolites that might help us tide over this crisis are greatly expected. A recently adopted method to evaluate the potentiation of metabolites is the plate-counting test. However, the method is time-consuming, strenuous, and unfeasible for a large scale of screening. A minimum inhibitory concentration (MIC) test by using a microtitre plate dilution method is convenient and economic for a large scale of identification, but it cannot be used to detect the potentiation. Here, the microtitre plate dilution method was modified to develop a novel test for evaluating metabolites that enable the killing of bacterial pathogens by antibiotics, designed as minimum killing concentration (MKC). To do this, bacterial number, incubation time, ionic strength of M9 medium, and inosine concentration are optimized using Escherichia coli. Different from the MIC test, which uses 5 × 104 CFU cells and performed in LB medium, the MKC test needed 1 × 107 CFU - 2 × 107 CFU cells and was carried out in M9 medium. Moreover, MKC test was suitable for bactericidal antibiotics such as cephalosporins, penicillins and carbapenems and was proportional to the plate-counting test. The developed MKC test was feasible for different metabolites and clinically multidrug-resistant pathogens, and measurement of minimum bactericidal concentration (MBC). Therefore, the MKC test was developed to accelerate the identification of compounds that promote antibiotic-mediated killing efficacy.

Inactivation of Nitrite-Dependent Nitric Oxide Biosynthesis Is Responsible for Overlapped Antibiotic Resistance between Naturally and Artificially Evolved Pseudomonas aeruginosa.

Metabolic flexibility of Pseudomonas aeruginosa could lead to new strategies to combat bacterial infection. The present study used gas chromatography-mass spectrometry (GC-MS)-based metabolomics to investigate global metabolism in naturally and artificially evolved strains with cefoperazone-sulbactam (SCF) resistance (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). Inactivation of the pyruvate cycle and nitric oxide (NO) biosynthesis was identified as characteristic features of SCF resistance in both evolved strains. Nitrite-dependent NO biosynthesis instead of an arginine-dependent NO pathway is responsible for the reduced NO, which is attributed to lower nitrite and electrons from the oxidation of NADH to NAD+ provided by the pyruvate cycle. Exogenous fumarate, NADH, nitrate, and nitrite promoted the NO level and thereby potentiated SCF-mediated killing. Unexpectedly, fumarate caused the elevation of nitrite, while nitrite/nitrate resulted in the increase of Cyt bc1 complex (providing electrons). These interesting findings indicate that the nitrite-dependent NO biosynthesis and the pyruvate cycle are mutual to promote NO metabolism. In addition, the NO-potentiated sensitivity to SCF was validated by NO donor sodium nitroprusside. These results reveal an endogenous NO-mediated SCF resistance and develop its reversion by metabolites in P. aeruginosa. IMPORTANCE Infections with Pseudomonas aeruginosa have become a real concern among hospital-acquired infections, especially in cystic fibrosis patients and immunocompromised individuals. Control of the pathogen is challenging due to antibiotic resistance. Since bacterial metabolic state impacts sensitivity and resistance to antibiotics, exploring and disclosing bacterial metabolic mechanisms can be used to develop a metabolome-reprogramming approach to elevate bacterial sensitivity to antibiotics. Therefore, GC-MS-based metabolomics is used to explore the similarities and differences of metabolomes between naturally and artificially evolved cefoperazone-sulbactam (SCF)-resistant P. aeruginosa (AP-RCLIN-EVO and AP-RLAB-EVO, respectively) from the same parent strain (AP-RCLIN). It identifies the depressed nitrite-dependent nitric oxide (NO) biosynthesis as the most overlapping characteristic feature between AP-RCLIN-EVO and AP-RLAB-EVO. This is because the pyruvate cycle fluctuates, thereby generating fewer NADH and providing fewer electrons for nitrite-dependent NO biosynthesis than the control. Interestingly, exogenous fumarate, NADH, nitrate, and nitrite as well as NO donor sodium nitroprusside promote NO generation to elevate sensitivity to SCF. These results highlight the way to understand metabolic mechanisms of antibiotic resistance and explore metabolic modulation to combat the bacterial pathogen.

A susceptibility locus at chromosome 3p21 linked to familial nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) poses one of the serious health problems in southern Chinese, with an incidence rate ranging from 15 to 50/100,000. Chromosome translocation t(1;3) and frequent loss of heterogeneity on short arms of chromosome 3 and 9 have been reported to be associated with NPC, and a genome-wide scan identified an NPC susceptibility locus on chromosome 4p15.1-q12 recently. In our study, we collected samples from 18 families at high risk of NPC from the Hunan province in southern China, genotyped with a panel of polymorphic markers on short arms of chromosomes 3, 9, and 4p15.1-q12. A locus on 3p21 was identified to link to NPC with a maximum logarithm of odds for linkage score of 4.18. Fine mapping located the locus to a 13.6-cM region on 3p21.31-21.2, where a tumor suppressor gene cluster resided. Our findings identified a novel locus for NPC and provided a map location for susceptibility genes candidates. In contrast to a recent study, no significant evidence for NPC linkage to chromosomes 4 and 9 was observed.

Effects of bone marrow-derived mesenchymal stem cells transplanted via the portal vein or tail vein on liver injury in rats with liver cirrhosis.

The aim of the present study was to compare the effects of bone marrow-derived mesenchymal stem cells (BMSCs) transplanted via the portal vein or tail vein on liver injury in rats with liver cirrhosis. BMSCs were isolated from rat bone marrow and labeled with green fluorescent protein (GFP). Then, the labeled BMSCs were injected into rats with liver injury via the portal vein or tail vein. Two weeks after transplantation, three rats in each group were sacrificed to test the distribution of GFP in the liver and the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin. Six weeks later, the remaining rats were sacrificed, and serum ALT, AST, albumin, hyaluronic acid (HA), laminin (LN) and procollagen type III (PC-III) levels were measured. The expression of albumin in the liver was analyzed by immunohistochemistry. Two weeks after BMSC transplantation, GFP-positive cells were detected in the livers of rats with BMSCs transplanted via the portal vein and tail vein. Compared with pre-transplantation levels, the ALT levels of the groups with BMSC transplantation via the portal vein and tail vein were significantly decreased after two and six weeks of BMSC transplantation (P<0.05), whereas the AST and albumin levels were not significantly different at two weeks after BMSC transplantation in the two groups (all P>0.05). However, the AST and albumin levels were significantly reduced at six weeks after BMSC transplantation (all P<0.05). At six weeks after BMSC transplantation, the serum HA, LN and PC-III levels in rats transplanted with BMSCs via the portal vein or tail vein had decreased significantly (all P<0.05), as compared with the levels prior to BMSC transplantation. BMSCs transplanted via the portal vein and tail vein achieved similar improvements in liver function in rats with liver cirrhosis, which suggests that peripheral venous administration is a convenient and effective route for BMSC transplantation.

[In vitro gene transfection by magnetic iron oxide nanoparticles and magnetic field increases transfection efficiency].

OBJECTIVE: To evaluate the feasibility of using iron oxide nanoparticles as gene vector and the effect of magnetic field on efficiency of transfection. METHODS: Iron oxide nanoparticles were prepared by alkaline precipitation of divalent and trivalent iron chloride. The surface of iron oxide nanoparticles was modified by self-assembled poly-L-lysine to form particle complexes (IONP-PLL). Transfection was determined by delivering reporter gene, PGL2-control encoding luciferase, to different cell lines using IONP-PLL as vector. The effect of magnetic field on efficiency of transfection was determined using Nd-Fe-B permanent magnet. RESULTS: Foreign gene could be delivered to various cell lines by IONP-PLL and expressed with high efficiency, but the transfection efficiency and time course varied in the different cell lines studied. Magnetic field could enhance the efficiency of transfection by 5 - 10 fold. CONCLUSION: IONP-PLL can be used as a novel non-viral gene vector in vitro, which offers a basis for gene delivery in vivo.

[Polymorphism of two novel SNPs, which locate on chromosome 9p21-22, in Han Chinese of Hunan].

OBJECTIVE: To search novel SNPs in exons and regulatory regions of CDKN2A and two novel putative tumor suppressor genes NGX6 and UBAP1, which all reside on chromosome 9p21-22. METHODS: The exons and regulatory regions of those genes were amplified and sequenced in 96 subjects. RESULTS: Two novel SNPs were found, one resides on the sixth exon of UBAP1 gene and the other on the fourth exon of CDKN2A gene. Two novel SNPs were submitted to the dbSNP database, and their access ID are rs3135929 and rs3088440. The polymorphic information contents of them are 0.102 and 0.213 respectively. There is linkage equilibrium between them, and the polymorphic information content of their haplotype is 0.302, higher than any of them individually. CONCLUSION: The polymorphic information content can be improved by using haplotype analysis of several SNPs.

Impact of experimental and demographic variables in serum peptide profiling based on magnetic bead and MALDI-TOF mass spectrometry.

BACKGROUND: Serum peptidome profile is a promising tool to identify physiologic or pathologic conditions. Stable serum peptidome profiles with high quality are essential for serum peptidome research. The aim of this study is to examine the impact of experimental and demographic variables in serum peptide profiling. METHODS: Magnetic bead combined with MALDI-TOF mass spectrometry was performed to evaluate the efficacy of various variables including the treatment of blood, the pretreatment of serum (magnetic beads and ultrafiltrate centrifugal filters), the mass spectrometry and the data handling. The influence of age and gender on serum peptidome was also analyzed in 123 healthy volunteers. RESULTS: The results showed that the sampling processing procedures were crucial for the serum peptidome profiles. There were obvious differences on the serum peptidome profiles between the age groups younger and older than 30. There was no difference between gender groups. CONCLUSIONS: A number of optimized and standardized variables should be defined in serum peptidome research based on magnetic beads and MALDI-TOF mass spectrometry. An extremely strict standard procedure and considerate arrangement should be applied.

Expression of LRRC4 has the potential to decrease the growth rate and tumorigenesis of glioblastoma cell line U251.

BACKGROUND & OBJECTIVE: LRRC4 is a novel gene that the author has identified recently, which displayed significant downregulation in primary brain tumor biopsies. This study was designed to investigate if LRRC4 has the potential of suppressing brain tumor growth. METHODS: The full-length coding region of LRRC4 gene was subcloned into the expression vector pcDNA3.1, the recombinant was introduced into the glioblastoma cell line U251 by liposome transfection, and the U251 cells stably expressing LRRC4 gene were established by G418 selection. Furthermore, cell proliferation assay, soft agar assay, tumorigenesis assay were taken to examine the effect of LRRC4 expression on cell growth and tumor formation. RESULTS: U251 cells stably expressing full-length coding region of LRRC4 were established by lipofection-mediated transfection and selected for further study. Compared with the nontransfected and vector-transfected cells, the cells transfected with LRRC4 cDNA exhibited a significant increase of expression of LRRC4 mRNA by Northern blot analysis. Further, when cell proliferation was followed over several days, the cells expressing the transfected LRRC4 cDNA grew more slowly than nontransfected cells. Consistently, the cells transfected with LRRC4 exhibited markedly lower colony formation rate. These clones were injected into athymic nude mice who was killed after 40 days and the tumor sizes were evaluated. Tumor volume in mice was significantly smaller in the group of cells stably transfected with LRRC4 cDNA than in the control. CONCLUSION: LRRC4 gene may be transfected into the human glioblastoma cell line U251. The expression of LRRC4 in U251 cells may have the potential to suppress tumor cell growth and the tumorigenesis of U251 cell transplanted in nude mice.

[Expression of connexin43 and connexin45 in nasopharyngeal carcinoma].

BACKGROUND AND OBJECTIVE: The gap junction plays an important role in the exchange of nutrients, ions, and regulatory molecules between cells. It will result in an uncontrolled cell proliferation if it is abnormal. It was reported that some cancer tissues and cancer cells had abnormal gap junction and restoration of the gap junction function could revert the cancer cells to a normal phenotype. Therefore, to investigate the expression of connexin(cx) in human nasopharyngeal carcinoma, may provide a new way for clinical diagnosis and the pathogenesis of nasopharyngeal carcinoma. METHOD: The expressions of Cx43 and Cx45 in the biopsies of nasopharyngeal carcinoma(NPC) and chronic nasopharyngitis tissues were determined by using the immunohistochemical staining. RESULTS: 1. Cx43 and Cx45 expressed differentially in NPC and chronic nasopharyngitis tissues(P < 0.01). In NPC, expression of Cx43 and Cx45 were 44.8% and 46.6%, respectively, while in columnar cells of chronic nasopharyngitis tissues, their expressions were 86.5% and 100% respectively, which were higher than those in NPC, 2. The percentage in squamous epithelial cells of chronic nasopharyngitis tissues was 29.7%, which was lower than that in NPC, (56.9%). 3. The expressions of Cx43 and Cx45 in paracancer columnar epithelial cells were lower than that in paracancer squamous epithelial cells (P < 0.001), but higher than that in NPC cells (P < 0.01). CONCLUSIONS: The abnormal expression of Cx43 and Cx45 in nasopharynx tissues may be associated with cancerization and squamatization of human nasopharynx tissue.

[Analysis of splicing variants in NASG 3'UTR, down-regulated in nasopharyngeal carcinoma, and its expression in multiple cancer tissues].

BACKGROUND & OBJECTIVE: NASG gene, a tissue-specific gene of human nasopharyngeal epithelium was isolated by suppression subtractive hybridization. This study was designed to analyze splicing variants in NASG 3'untranslated region (UTR) and its expression profiling in multiple cancer tissues. METHODS: The PCR primers were designed in NASG 3'UTR around the splicing variants and reverse transcription-polymerase chain reaction (RT-PCR) was performed. The PCR products were separated and sequenced. The expression patterns of NASG were detected by RT-PCR among nasopharyngeal carcinoma (NPC) cell line HNE1, primary human embryo nasopharyngeal epithelial cells, NPC biopsies, and normal adult nasopharyngeal epithelial tissues. Its expression profiling in multiple cancer tissues were tested by cancer profiling array hybridization. RESULTS: There were three splicing variants in NASG 3'UTR. NASG was identified to be down-regulated in NPC cell line HNE1 and 71% of the NPC biopsies, but up-regulated in 25% lung of the cancer biopsies, and not express in other cancer tissues and normal tissues. CONCLUSION: There were three splicing variants in NASG 3'UTR. Its abnormal expression may be an important molecular event in NPC and lung cancer.

Poly(L-lysine)-modified silica nanoparticles for the delivery of antisense oligonucleotides.

Silica nanoparticles were prepared in a microemulsion system, using polyoxyethylene nonylphenyl ether/cyclohexane/ammonium hydroxide. The surface charge of the particle was modified with PLL [poly(L-lysine)]. PAGE demonstrated the ability of PMS-NP (PLL-modified silica nanoparticles) to bind and protect antisense ODNs (oligonucleotides). The intracellular localization of FITC-labelled ODN was investigated by fluorescence microscopy. The results demonstrated that ODN could be delivered to cytoplasm. Flow-cytometry analysis showed a 20-fold enhancement of ODN delivered by PMS-NP compared with free ODN for a serum-free medium. Blocking efficacy of c- myc antisense ODN, delivered by PMS-NP, was examined in HNE1 and HeLa cell lines. Significant down-regulation of c- myc mRNA levels was observed in both the cell lines. However, the cellular uptake efficiency and antisense effects on target gene decreased in the presence of serum-containing medium. The analysis of the filtration assay showed that PMS-NP interacted with serum proteins. These results indicated that PMS-NP was a suitable delivery vector for antisense ODN, although its delivery efficiency decreased in the presence of a serum-containing medium.

IONP-PLL: a novel non-viral vector for efficient gene delivery.

BACKGROUND: Non-viral methods of gene delivery have been an attractive alternative to virus-based gene therapy. However, the vectors that are currently available have drawbacks limiting their therapeutic application. METHODS: We have developed a self-assembled non-viral gene carrier, poly-L-lysine modified iron oxide nanoparticles (IONP-PLL), which is formed by modifying poly-L-lysine to the surface of iron oxide nanoparticles. The ability of IONP-PLL to bind DNA was determined by ratio-dependent retardation of DNA in the agarose gel and co-sedimentation assay. In vitro cytotoxic effects were quantified by MTT assay. The transfection efficiency in vitro was evaluated by delivering exogenous DNA to different cell lines using IONP-PLL. Intravenous injection of IONP-PLL/DNA complexes into mice was evaluated as a gene delivery system for gene therapy. The PGL2-control gene encoding firefly luciferase and the EGFP-C2 gene encoding green fluorescent protein were used as marker genes. RESULTS: IONP-PLL could bind and protect DNA. In contrast to PLL and cationic liposomes, IONP-PLL described here was less cytotoxic in a broad range of concentrations. In the current study, we have demonstrated that IONP-PLL can deliver exogenous gene to cells in vitro and in vivo. After intravenous injection, IONP-PLL transferred reporter gene EGFP-C2 to lung, brain, spleen and kidney. Furthermore, we have demonstrated that IONP-PLL transferred exogenous DNA across the blood-brain barrier to the glial cells and neuron of brain. CONCLUSIONS: IONP-PLL, a low-toxicity vector, appears to have potential for fundamental research and genetic therapy in vitro and in vivo, especially for gene therapy of CNS disease.

[Biocompatibility of poly-l-lysine-modified silica nanoparticles].

BACKGROUND & OBJECTIVE: Poly-l-lysine-modified silica nanoparticle(PMS-NP) was a novel non-viral vector for gene delivery. The current study was designed to evaluate the biocompatibility of PMS-NP for its further utilization in vivo. METHODS: Cell transfection and flow cytometry were used to elucidate the delivery efficiency of plasmid DNA and antisense ODN mediated by PMS-NP in the presence of serum-containing medium. Subsequently, the biocompatibility of PMS-NP in vivo was evaluated using filtration assay of plasma proteins and erythrocyte aggregation assay. RESULTS: The abilities of PMS-NP to deliver plasmid DNA and antisense ODN in vitro clearly decreased in the presence of serum-containing medium. PMS-NP/DNA(ODN)complexes bound plasma proteins and triggered erythrocyte aggregation. CONCLUSION: PMS-NP might interact with plasma proteins, resulting in decreased transfection efficiency in vitro. And filtration assay of plasma proteins and the erythrocyte aggregation assay demonstrated that the interaction of PMS-NP with plasma proteins and erythrocytes might play a negative role in gene transfection efficiency in vivo. And its biocompatibility needs to be further improved.