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Biomedical subjects

Jun Fu

Publications and source records attributed to Jun Fu.

At least 19 recordsLinked to original sources
PubMed2026

Development of a new recombineering system for Edwardsiella species.

Edwardsiella species are important aquaculture pathogens that also cause opportunistic infections in humans, necessitating efficient genome editing tools to study their pathogenesis and develop control strategies. In this study, we identified and characterized six endogenous recombinases pairs from Edwardsiella and its phages. Among these, the BAS_MS17 system exhibited the highest recombination efficiency in E. piscicida EIB202Δp. Extending homology arms from 150 bp to 200 bp improved editing efficiency by 2-fold, while the addition of Redg or Plug further enhanced recombination by 3-fold and 2.5-fold, respectively, without compromising accuracy (100%). More importantly, when applied to E. piscicida sdu12S, Redg or Plug improved the editing efficiency by 8-fold and 7-fold, respectively. Deletion of the phage-derived single-strand binding protein (SSB) reduced efficiency to 25% of the BAS_MS17 level, whereas expression of the endogenous RecA-family SSB (rSSB) increased recombinant yield by 5-fold, highlighting functional conservation. Furthermore, SSB proteins from heterologous hosts failed to enhance recombination efficiency. Using the optimized system, we successfully knocked out ten distinct genes, including virulence-associated loci, with editing accuracy exceeding 85%. Phenotypic analysis revealed that luxR, but not the other tested genes, contributes to biofilm formation. Virulence evaluation results showed that aroA, fur, and hfq are critical virulence-associated factors. Collectively, this streamlined recombineering system provides a simple, rapid, and efficient genetic tool for Edwardsiella, supporting mechanistic studies of virulence and the development of live attenuated vaccine candidates.

Edwardsiella piscicida↗
PubMed2006

Reconstitution of the myxothiazol biosynthetic gene cluster by Red/ET recombination and heterologous expression in Myxococcus xanthus.

Although many secondary metabolites exhibiting important pharmaceutical and agrochemical activities have been isolated from myxobacteria, most of these microorganisms remain difficult to handle genetically. To utilize their metabolic potential, heterologous expression methodologies are currently being developed. Here, the Red/ET recombination technology was used to perform all required gene cluster engineering steps in Escherichia coli prior to the transfer into the chromosome of the heterologous host. We describe the integration of the complete 57-kbp myxothiazol biosynthetic gene cluster reconstituted from two cosmids from a cosmid library of the myxobacterium Stigmatella aurantiaca DW4-3/1 into the chromosome of the thus far best-characterized myxobacterium, Myxococcus xanthus, in one step. The successful integration and expression of the myxothiazol biosynthetic genes in M. xanthus results in the production of myxothiazol in yields comparable to the natural producer strain.

Bacterial Proteins↗
PubMed2006

The landscape pattern characteristics of coastal wetlands in Jiaozhou Bay under the impact of human activities.

In this study, we interpreted coastal wetland types from an ASTER satellite image in 2002, and then compared the results with the land-use status of coastal wetlands in 1952 to determine the wetland loss and degradation around Jiaozhou Bay. Seven types of wetland landscape were classified, namely: shallow open water, inter-tidal flats, estuarine water, brackish marshes, salt ponds, fishery ponds and ports. Several landscape pattern indices were analysed: the results indicate that the coastal wetlands have been seriously degraded. More and more natural wetlands have been transformed into artificial wetlands, which covered about 33.7% of the total wetlands in 2002. In addition, we used a defined model to assess the impacts of human activities on coastal wetlands. The results obtained show that the coastal wetlands of Jiaozhou Bay have suffered severe human disturbance. Effective coastal management and control is therefore needed to solve the issues of the coastal wetland loss and degradation existing in this area.

Biodiversity↗
PubMed2006

Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts.

The present study was designed to determine the effects of fluid shear stress on the mRNA expression of carbonic anhydrase II (CAII) in polarized rat osteoclasts. Cellular morphology of the polarized osteoclasts generated by a mechanical anatomical technique was examined by tartrate-resistant acid phosphatase (TRAP) staining and the osteoclastic resorption of dentine slices. The polarized osteoclasts were then stress-loaded by using a flow shear stress device newly developed by the osteoclast research group (patent number 200420034438; China), at 9 dyne/cm(2) for various time periods [0 (control group), 15, 30, 60, and 120 min], or at various stress levels [0 (control), 0.9, 2.9, 8.7, and 26.3 dyne/cm(2)] for 30 min. The mRNA expression of CAII was quantified using real-time fluorescent quantitative PCR (RT-PCR) and the data were analyzed with SPSS 12.0 software. The polarized osteoclasts were larger than regular monocytes (about 30 microm diameter) with irregular configuration, and the majority of polarized osteoclasts appeared to be spherical and had approximately 2-20 nuclei. The TRAP positive polarized osteoclasts showed asymmetrical red staining in the cytoplasm, and had many filaments and vacuoles. These cells formed resorptive pits in dentine slices. The levels of CAII mRNA expression were shown to be time-dependent, with the E+5 copy numbers being 7.88+/-0.09, 11.14+/-0.12, 15.83+/-0.18, 1.94+/-0.02, and 1.37+/-0.01 in cells treated at 9 dyne/cm(2) for 0, 15, 30, 60 and 120 min, respectively (P < 0.05). The levels of CAII mRNA expression (E+5 copy numbers) in cells treated with the stress levels of 0, 0.9, 2.9, 8.7 and 26.3 dyne/cm(2) were 7.97+/-0.201, 11.26+/-0.688, 15.94+/-0.201, 31.88+/-1.496, and 45.08+/-2.639, respectively (P < 0.05). These results indicate that there is a relationship between the fluid shear stress and the mRNA expression of CAII in polarized rat osteoclasts.

Animals↗
PubMed2006

Recombineering reagents for improved inducible expression and selection marker re-use in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe is an excellent model organism for cell biology. However, its genetic toolbox is less developed than that of Saccharomyces cerevisiae. In the first part of this study we describe an improved inducible expression vector based on tetracycline regulation of the CaMV35S promoter, which is also capable of chromosomal integration and therefore works in minimal and in rich media. We found that anhydrotetracycline is a superior ligand for induction. Maximum expression levels were observed after 12 h in minimal media (EMM) and after 9 h in rich media (YES), which is faster than the nmt1 promoter system. The system was combined with a convenient recombineering-based subcloning strategy for ease of cloning. In the second part we present four template plasmids, pSVEM-bsd, pSVEM-nat, pSVEM-kan and pSVEM-hph, which harbour four recyclable disruption cassettes based on the Cre recombinase lox71/66 strategy for use in PCR targeting methods. Cre-mediated excision leaves a non-functional mutant lox site in the genome, allowing the reiterative usage of these cassettes for multiple targetings. These cassettes are also configured with dual eukaryotic/prokaryotic promoters so that they can be used for recombineering in E. coli. Amongst other purposes, this permits the rapid and convenient creation of targeting constructs with much longer homology arms for difficult and complex targetings in the Sz. pombe genome.

Escherichia coli↗
PubMed2006

Suppression of primary breast, colon, gastric and bladder cancers cell growth in vitro by CKBM, a natural product.

CKBM is a product composed of natural ingredients and had been shown to possess certain anti-cancer effects in vitro and in vivo. The aim of the present study is to analyze the chemosensitivity in the treatment of primary colon, breast, gastric and bladder cancer cells by CKBM. A total of 77 patients with cancers of breast, colon, stomach or bladder were included in the present study. Primary cancer cells were isolated from the surgical removed tumors and treated with various dosages of CKBM for 5 days. ATP is then extracted and measured by luminescence assay. CKBM treatment inhibited primary colon, breast, gastric and bladder cancer growth dose-dependently. The IC values were smaller from tumor cells at early stages, when compared with the ones at later stages. The present study strongly indicated that CKBM exerted cytotoxic effect on primary cancer cells.

Adolescent↗
PubMed2006

Metabolic engineering of Pseudomonas putida for methylmalonyl-CoA biosynthesis to enable complex heterologous secondary metabolite formation.

An operon consisting of three open reading frames, annotated in silico as methylmalonyl-CoA (mm-CoA) epimerase, mm-CoA mutase (MCM), and meaB, was identified in the sequencing project of the myxobacterium Sorangium cellulosum So ce56. This putative MCM pathway operon was subcloned from a bacterial artificial chromosome by Red/ET recombineering onto a minimal replicon derived from p15A. This plasmid was modified for integration and heterologous expression in Pseudomonas putida to enable the production of complex secondary metabolites requiring mm-CoA as precursor. Methylmalonate was identified in the recombinant P. putida strain by an analysis method based on gas chromatography/mass spectrometry. The engineered strain is able to synthesize polyketides requiring mm-CoA as an extender unit, which was demonstrated by the production of myxothiazol after integration of the biosynthetic gene cluster into the chromosome, followed by induction of expression.

Acyl Coenzyme A↗
PubMed2006

Displacement-based binning of time-dependent computed tomography image data sets.

Respiration can cause tumors in the thorax or abdomen to move by as much as 3 cm; this movement can adversely affect the planning and delivery of radiation treatment. Several techniques have been used to compensate for respiratory motion, but all have shortcomings. Manufacturers of computed tomography (CT) equipment have recently used a technique developed for cardiac CT imaging to track respiratory-induced anatomical motion and to sort images according to the phase of the respiratory cycle they represent. Here we propose a method of generating CT images that accounts for respiratory-induced anatomical motion on the basis of displacement, i.e., displacement-binned CT image sets. This technique has shown great promise, however, it is not fully supported by currently used CT image reconstruction software. As an interim solution, we have developed a method for extracting displacement-binned CT image data sets from data sets assembled on the basis of a prospectively determined breathing phase acquired on a multislice helical CT scanner. First, the projection data set acquired from the CT scanner was binned at small phase intervals before reconstruction. The manufacturer's software then generated image sets identified as belonging to particular phases of the respiratory cycle. All images were then individually correlated to the displacement of an external fiducial marker. Next, CT image data sets were resorted on the basis of the displacement and assigned an appropriate phase. Finally, displacement-binned image data sets were transferred to a treatment-planning system for analysis. Although the technique is currently limited by the phase intervals allowed by the CT software, some improvement in image reconstruction was seen, indicating that this technique is useful at least as an interim measure.

Algorithms↗
PubMed2006

An improved recombineering approach by adding RecA to lambda Red recombination.

Recombineering is the use of homologous recombination in Escherichia coli for DNA engineering. Of several approaches, use of the lambda phage Red operon is emerging as the most reliable and flexible. The Red operon includes three components: Redalpha, a 5' to 3' exonuclease, Redbeta, an annealing protein, and Redgamma, an inhibitor of the major E. coli exonuclease and recombination complex, RecBCD. Most E. coli cloning hosts are recA deficient to eliminate recombination and therefore enhance the stability of cloned DNAs. However, loss of RecA also impairs general cellular integrity. Here we report that transient RecA co-expression enhances the total number of successful recombinations in bacterial artificial chromosomes (BACs), mostly because the E. coli host is more able to survive the stresses of DNA transformation procedures. We combined this practical improvement with the advantages of a temperature-sensitive version of the low copy pSC101 plasmid to develop a protocol that is convenient and more efficient than any recombineering procedure, for use of either double- or single-stranded DNA, published to date.

Animals↗
PubMed2006

[Bevacizumab (Avastin) inhibits lung cancer-induced pulmonary microvascular angiogenesis].

OBJECTIVE: To study the effect of bevacizumab (Avastin) on pulmonary microvascular angiogenesis induced by lung cancer. METHODS: The effects of Avastin on cell growth, cell cycle and apoptosis of lung microvascular endothelial cells were assayed by flow cytometry. RESULTS: Avastin inhibited the growth of lung microvascular endothelial cells and induced increase of cell apoptosis index in a concentration-dependent manner. With the increment of Avastin concentration from 25 to 100 microg/ml, avascular area increased from 0.944-/+0.073 cm square to 5.189-/+0.192 cm square, and the cell apoptosis index increased from 32.5%-/+1.5% to 39.25-/+1.6%. CONCLUSION: Avastin can inhibit the growth and induce apoptosis of lung microvascular endothelial cells in three-dimensional culture, possibly in association with cell cycle arrest in G(0/1) phase.

Adenocarcinoma↗
PubMed2006

[Effects of hyperthermic chemotherapy on gastrointestinal cancer cells in vitro].

BACKGROUND & OBJECTIVE: Little evidence is known from experimental research for intraoperative hyperthermic intraperitoneal chemotherapy. This study was to investigate the effect of hyperthermic chemotherapy on gastrointestinal cancer cells in vitro and explore the possible factors which may affect this method. METHODS: Gastric cancer cell line MGC-803 and intestinal cancer cell line HCT-116 were chosen. Cells were treated with different drugs, temperatures and duration. Cell viability and growth were measured by MTS-PMS assay. The morphology of the cells was observed under a microscope. RESULTS: Significant synergistic effect was observed when the two cancer cell lines were treated with 5-FU, MMC, DDP and THP in combination with elevated hyperthermia from 41 degrees C to 45 degrees C compared with control group (P<0.01). The strongest effect was achieved at 45 degrees C, which the inhibitory effects of these four drugs were 61.7%, 79.2%, 88.7%, 94.7% on MGC-803 and 76.4%, 78.7%, 77.8%, 91.7 on HCT-1116, respectively. The inhibitory effect demonstrated a time and dose-dependent manner in HCT-116 cells within a certain period of time. The effect revealed a flat curve after 90 min when HCT-116 was treated with 43 centigrade. THP had the strongest effect at any conditions among all tested drugs (P<0.01). Either simple thermotherapy or chemohyperthermia displayed considerable killing effects on cancer cells which were confirmed by microscope observation. And a great deal of dead cells were observed when treated with chemohyperthermia. CONCLUSIONS: DDP or MMC reveals relatively satisfactory antitumor effects with the optimal temperature of 43-45 degrees C. Taken practical application into consideration, 60 min may be selected in clinical use. Synergistic antitumor effects of THP in combination with hyperthermia were prior to 5-FU, DDP or MMC, which deserve further clinical research.

Antibiotics, Antineoplastic↗
PubMed2006

[Isolation of bone marrow-derived rat osteoclast-like cells with the digestion of trypsin].

OBJECTIVE: To obtain highly enriched osteoclasts in vitro. METHODS: The bone marrow cells of 1-day, 12-day, 24-day and 36-day old Sprague-Dawley (SD) rats were separately cultured with the presence of 1,25-(OH)2D3 and dexamethasone, and the osteoclast-like cells were identified by Trap staining and scanning electron microscope observation and were purified by trypsin digestion. The count of Trap positive osteoclast-like cells was analyzed statistically. RESULTS: The osteoclast-like cells of each group were Trap positive cells and could form the bone absorption lacunas in vitro. The amounts of osteoclast-like cells were different statistically between the groups (P < 0.05) except between the groups of 1-day old and 12-day old (P > 0.05). Highly enriched osteoclast-like cells were harvested by the digestion of 0.25% trypsin and 0.02% EDTA. CONCLUSION: It's indicated that a large amount of highly enriched osteoclast-like cells could be obtained through the culture of bone marrow cells of 10-day and 12-day old SD rats with 1,25-(OH)2D3 and dexamethasone and the digestion of trypsin/EDTA.

Animals↗
PubMed2006

[Effects of pirarubicin chemotherapy combined with hyperthermia on gastric cancer in vitro].

OBJECTIVE: To evaluate the effect of pirarubicin (THP) in combination with hyperthermia on gastric cancer tissues in vitro and explore the underlying mechanisms. METHODS: In vitro three-dimensional culture models were established with tissue biopsies from 36 patients with pathologically confirmed gastric cancer. The tumor cell viability was measured by MTS-PMS assay, and HE staining was used to study the histomorphological changes of the tissues following chemotherapy and hyperthermia. RESULTS: Synergistic tumor cell-killing effects of cisplatin, THP, and mitomycin with hyperthermia was observed in the tumor tissues (P=0.000), and THP exhibited stronger cytotoxic effects than the other drugs. Histomorphological study suggested strong killing effects of THP on the tumor tissues, which displayed disrupted tissue structure, cellular degradation and necrosis, karyopyknosis and karyolysis, with cytoplasm loss. The anti-tumor effects of THP were associated with clinical staging and pathological grading of the tumors (P=0.000), but not with the patients' gender, age, tumor size and preoperative CEA levels (P>0.05). CONCLUSION: Pirarubicin shows good synergistic effects with hyperthermia, and the cytotoxicity of pirarubicin against gastric cancer tissue is enhanced considerably by mild hyperthermia. THP can be a potential therapeutic drug for intraperitoneal chemohyperthermia.

Adenocarcinoma↗
PubMed2005

Molecular motions of different scales at thin polystyrene film surface by lateral force microscopy.

Lateral force microscopy (LFM) was used to probe the molecular motions at thin polystyrene film surface. The effect of the applied load on the LFM measurements was investigated by presenting both the LFM results and the surface morphology after several scans over the same area. Depending on the loads, the scanning can be nonperturbative (without alternating the surface morphology) or perturbative (patterning the surface). Temperature-dependent LFM measurements were conducted in order to determine the apparent transitions at the surface. Perturbative scans under high loads (e.g., 150 nN) witnessed that the apparent transitions shifted to low temperatures with an increasing scan rate, while the transitions behaved oppositely under lower loads (1, 10, and 20 nN). The heating effect is suggested to account for the behavior under high loads. According to our results from nonperturbative LFM, the apparent glass transition temperature (T(g)s) is more than 10 K lower than the bulk value. Moreover, rate-dependent LFM measurements were performed under 1 nN in order to detect the surface molecular motions. Time-temperature superposition yields a master curve exhibiting three apparent relaxation peaks. The molecular motions at the surface are discussed on the context of the coupling model.

Journal Article↗
PubMed2005

Ordered honeycomb-structured gold nanoparticle films with changeable pore morphology: from circle to ellipse.

Two-dimensionally ordered honeycomb structures have been prepared on dodecanethiol-capped gold nanoparticle films by blowing moist air across the surface of the nanoparticle solution. The pore morphology can be altered from circle to ellipse with tunable aspect ratios by carefully controlling the direction and velocity of airflow. The formation mechanisms of different surface morphologies have been discussed in terms of the surface and interfacial tension.

Journal Article↗
PubMed2005

Heterologous expression of a myxobacterial natural products assembly line in pseudomonads via red/ET recombineering.

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.

DNA, Recombinant↗
PubMed2005

[Correlation between ATP bioluminescence tumor chemosensitivity assay and clinical response in ovarian cancer].

OBJECTIVE: To determine the correlation between results of ATP bioluminescence tumor chemosensitivity assay (ATP-TCA) of human ovarian cancer specimens in vitro and clinical chemo-therapeutic responses of patients. METHODS: Thirty-four freshly taken ovarian cancer specimens (28 cases) and ascites (6 cases) and 9 chemotherapeutic drugs were tested in vitro for cancer chemosensitivity by ATP-TCA. RESULTS: Among the 34 ovarian cancer cases, the efficacy of ATP-TCA is 94.0%, the sensitivity, the specificity, the positive and negative predicting values, and an overall predicting value in vitro and vivo were 90.0%, 91.7%, 94.7%, 84.6% and 90.6%, respectively. CONCLUSION: The results of ATP-TCA assay are correlated well with clinical treatment responses. The assay may be an important and useful method for individual-based chemotherapy of cancers.

Adenosine Triphosphate↗