SrfABC Toxin from Xenorhabdus stockiae Induces Cytotoxicity and Apoptosis in HeLa Cells.

Our previous study showed that the srfABC operon, which was originally identified in Salmonella enterica as an SsrB-regulated operon clustered with the flagellar class 2 operon, exhibited significant cytotoxicity against insect midgut CF-203 cells and injectable insecticidal activity against Helicoverpa armigera larvae. The srfABC operon was widely distributed among bacteria, which raises the question of their biological roles in different species. In this study, we investigated the cytotoxic effect of SrfABC toxin on mammalian cell lines. When simultaneously expressed in the Escherichia coli cytoplasm, SrfABC exhibited cytotoxicity against all tested mammalian cancer cell lines (B16, 4T-1, Hep-3B, and HeLa) in a dose-dependent manner. Intracellular expression of SrfA-FLAG, SrfB-FLAG, or SrfC-FLAG also resulted in inhibition of proliferation and apoptosis on HeLa cells. When incubated with HeLa cells separately, SrfA, SrfB, and SrfC proteins alone could enter HeLa cells, then induce apoptosis and cytotoxicity. SrfC protein shifts its localization from cytoplasm to nucleus with the aid of SrfA and/or SrfB protein. Although SrfA, SrfB, and SrfC proteins alone exhibited a cytotoxic effect against HeLa cells, all three components were essential for the full cytotoxicity. Native PAGE and co-immunoprecipitation assay demonstrated that SrfA, SrfB, and SrfC proteins could interact with each other and form a heteromeric complex.

Screening a fosmid library of Xenorhabdus stockiae HN_xs01 reveals SrfABC toxin that exhibits both cytotoxicity and injectable insecticidal activity.

Xenorhabdus spp., entomopathogenic bacteria symbiotically associated with the nematodes of the Steinernematid family, are known to produce several toxic proteins that interfere with the cellular immune responses of insects. In order to identify novel cytotoxins from Xenorhabdus spp., a fosmid library of X. stockiae HN_xs01 strain was constructed and the cytotoxicity of fosmid clones was tested against insect midgut CF-203 cells. An FS2 clone bearing the srfABC operon, originally identified in Salmonella enterica, exhibited excellent cytotoxicity against CF-203 cells. The srfABC operon alone exhibited cytotoxic effects and all three components of SrfABC toxin were essential for full cytotoxicity. Immunofluorescence studies showed that SrfABC toxin could depolymerize microtubules and disrupt mitochrondria. Flow cytometer analysis demonstrated that SrfABC toxin significantly induced G2/M phase arrest and apoptosis in CF-203 cells. Furthermore, SrfABC toxin exhibits highly injectable insecticidal activity against Helicoverpa armigera larvae. As is often found in host-associated microorganisms, SrfABC toxin is thought to play an important role in host colonization.

Solubility enhancement of Cry2Aa crystal through carboxy-terminal extension and synergism between the chimeric protein and Cry1Ac.

It was reported that the highly conserved C-terminal region of Bacillus thuringiensis Cry1A protoxins was very important for parasporal crystal formation and solubility feature in alkaline environment. In order to improve the solubilization efficiency of Cry2Aa crystal, the coding sequences of Cry2Aa protein and the C-terminal half of Cry1Ac were fused seamlessly through Red/ET homologous recombination and expressed in an acrystalliferous B. thuringiensis strain under the control of the cry1Ac promoter and terminator. Microscopic observation revealed that the recombinant strain containing the chimeric gene cry2Aa-1Ac produced distinct parasporal inclusion with semispherical to approximately cuboidal shape during sporulation. SDS-PAGE analysis showed that this strain expressed stable 130-kDa Cry2Aa-1Ac chimeric protein, which was confirmed to be the correctly expressed product by LC-MS/MS. The chimeric protein inclusion could be effectively dissolved at pH 10.5 and activated by trypsin like the parental Cry1Ac crystal. While, the parental Cry2Aa crystal exhibited very low solubility under this condition. Bioassays against third-instar larvae of Helicoverpa armigera proved that the chimeric protein was more toxic than Cry2Aa. Additionally, synergistic effect was clearly detected between the chimeric protein and Cry1Ac against H. armigera, while there was only additive effect for the combination of wild Cry2Aa and Cry1Ac. These results indicated that the developed chimeric protein might serve as a potent insecticidal toxin used in the field against lepidopteran pests.

The full-length Cry1Ac protoxin without proteolytic activation exhibits toxicity against insect cell line CF-203.

The new dual model for Bacillus thuringiensis insecticidal mechanism proposed that Cry1A protoxins without proteolytic activation could bind to insect midgut receptors to exert toxicity. To evaluate insecticidal potency of Cry1Ac protoxin at precluding interference of midgut proteases, the cytotoxicity of Cry1Ac protoxin against midgut cell line CF-203 derived from Choristoneura fumiferana was analyzed. It was revealed that Cry1Ac protoxin was toxic to CF-203 cells and there existed certain differences in the cytological changes when treated with protoxin and toxin. Our cell-based study provided direct evidence for the proposed dual model and shed light on exploring the difference between two toxic pathways elicited by intact protoxin and activated toxin.

"Cre/loxP plus BAC": a strategy for direct cloning of large DNA fragment and its applications in Photorhabdus luminescens and Agrobacterium tumefaciens.

Heterologous expression has been proven to be a valid strategy for elucidating the natural products produced by gene clusters uncovered by genome sequencing projects. Efforts have been made to efficiently clone gene clusters directly from genomic DNA and several approaches have been developed. Here, we present an alternative strategy based on the site-specific recombinase system Cre/loxP for direct cloning gene clusters. A type three secretion system (T3SS) gene cluster (~32 kb) from Photorhabdus luminescens TT01 and DNA fragment (~78 kb) containing the siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58 have been successfully cloned into pBeloBAC11 with "Cre/loxP plus BAC" strategy. Based on the fact that Cre/loxP system has successfully used for genomic engineering in a wide range of organisms, we believe that this strategy could be widely used for direct cloning of large DNA fragment.

A rifampicin-resistant (rpoB) mutation in Pseudomonas protegens Pf-5 strain leads to improved antifungal activity and elevated production of secondary metabolites.

Ribosome engineering has proven to be a practical method for increasing antibiotic production, and is extensively applied to strain improvement in antibiotic production and activation of silent genes in several prokaryotes. In this study, ribosome engineering was used to improve production of bioactive secondary metabolites produced by Pseudomonas protegens Pf-5. Rifampicin-resistant mutants that bear the H531N in the ß-subunit of RNA polymerase showed improved antifungal activity and morphological changes. The production of several secondary metabolites in R55 mutant was significantly improved using high-performance liquid chromatography (HPLC) analysis. Two antibiotics with antifungal activity, 2, 4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), which may contribute to the improved antifungal activity of the R55 mutant, were identified by mass spectrometer (MS) analysis.

The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis.

It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis.

[Experimental study of repairing full-thickness articular cartilage defect with chondrocyte-sodium alginate hydrogel-SIS complex].

OBJECTIVE: To explore the effect of tissue engineered cartilage reconstructed by using sodium alginate hydrogel and SIS complex as scaffold material and chondrocyte as seed cell on the repair of full-thickness articular cartilage defects. METHODS: SIS was prepared by custom-made machine and detergent-enzyme treatment. Full-thickness articular cartilage of loading surface of the humeral head and the femoral condyle obtained from 8 New Zealand white rabbits (2-3 weeks old) was used to culture chondrocytes in vitro. Rabbit chondrocytes at passage 4 cultured by conventional multiplication method were diluted by sodium alginate to (5-7) x 10(7) cells/mL, and then were coated on SIS to prepare chondrocyte-sodium alginate hydrogel-SIS complex. Forty 6-month-old clean grade New Zealand white rabbits weighing 3.0-3.5 kg were randomized into two groups according to different operative methods (n = 20 rabbits per group), and full-thickness cartilage defect model of the unilateral knee joint (right or left) was established in every rabbit. In experimental group, the complex was implanted into the defect layer by layer to construct tissue engineered cartilage, and SIS membrane was coated on the surface to fill the defect completely. While in control group, the cartilage defect was filled by sodium alginate hydrogel and was sutured after being coated with SIS membrane without seeding of chondrocyte. General condition of the rabbits after operation was observed. The rabbits in two groups were killed 1, 3, 5, 7, and 9 months after operation, and underwent gross and histology observation. RESULTS: Eight rabbits were excluded due to anesthesia death, wound infection and diarrhea death. Sixteen rabbits per group were included in the experiment, and 3, 3, 3, 3, and 4 rabbits from each group were randomly selected and killed 1, 3, 5, 7, and 9 months after operation, respectively. Gross observation and histology Masson trichrome staining: in the experimental group, SIS on the surface of the implant was fused with the host tissue, and the interface between them disappeared 1 month after operation; part of the implant was chondrified and the interface between the implant and the host tissue was fused 3 months after operation; the implant turned into fibrocartilage 5 months after operation; fiber arrangement of the cartilage in the implant was close to that of the host tissue 7 months after operation; cartilage fiber in the implant arranged disorderly and active cell metabolism and proliferation were evident 9 months after operation. While in the control group, no repair of the defect was observed 9 months after operation. No obvious repair was evident in the defects of the control group within 9 months after operation. Histomorphometric evaluation demonstrated that the staining intensity per unit area of the reparative tissue in the defect of the experimental group was significant higher than that of the control group at each time point (P < 0.05), the chondrification in the experimental group was increased gradually within 3, 5, and 7 months after operation (P < 0.05), and it was decreased 9 months after operation comparing with the value at 7 months after operation (P < 0.05). CONCLUSION: Constructed by chondrocyte-sodium alginate hydrogel-SIS in complex with surficial suturing of SIS membrane, the tissue engineered cartilage can in-situ repair cartilage defect, promote the regeneration of cartilage tissue, and is in line with physiological repair process of articular cartilage.

Effects of 20% demineralization on surface physical properties of compact bone scaffold and bone remodeling response at interface after orthotopic implantation.

To enhance osteointegration with preservation of mechanical strength, a surface modification technique using 20% surface demineralization in a controlled manner was applied to custom-built cylindrical bio-derived compact bones (20% surface-demineralized cylindrical compact bio-derived bone scaffold: SDCBS); an undemineralized version was the control. The micro-surface topography of the two types of bone scaffolds was characterized by atomic force microscopy (AFM) and scanning electron microscopy (SEM). 20% demineralization led to significant increases in surface roughness (38.19%, P=0.001) and surface area (15.1%, P=0.030), compared with the control group's, while the decrease in mechanical properties was not statistically significant. Results of orthotopic implantation for 9 months demonstrated that 20% surface demineralization caused significantly rapid and homogeneous bone remodeling at the interface compared to control and led to a significantly rapid osteointegration of SDCBS with the host bone at the early and intermediate stages of osteointegration. The study indicates the potential of SDCBS in repairing clinical bone defects, and would help direct the use of various processes of biomaterials to support defect repairs within osseous sites.

Variations in the ratios of co-cultured mesenchymal stem cells and chondrocytes regulate the expression of cartilaginous and osseous phenotype in alginate constructs.

As mesenchymal stem cells (MSCs) are capable of self-renewal and multilineage differentiation, the feasibility and efficacy of co-culturing human MSCs (hMSCs) with rabbit articular chondrocytes (rACs) to promote chondrogenic and osteogenic differentiation of hMSCs for clinical osteoarthritic therapy were investigated in the present study. The two distinct cell types were encapsulated in alginate hydrogels singly or in one of three ratios (2:1, 1:1, 1:2 of hMSCs to rACs) and cultured under chondrogenic conditions for 28 days. The results demonstrated that newly synthesized cartilaginous extracellular matrix (ECM) and type II collagen (col-2) gene signal were upregulated with greater hMSC ratios and longer culture periods. However, a specific col-2 gene probe for human was found only in single hMSC group but absent in all co-culture groups, which indicate that the enhanced cartilaginous phenotype originated from the co-cultured rACs. Osseous phenotype was histologically detected only in the 2:1 group on day 28; and xenogenic osteocalcin assay showed that it originated from hMSCs. This suggests that variations in the ratios of co-cultured hMSC and rAC regulated the cartilaginous and osseous phenotype as well as the differentiation of hMSCs in alginate constructs. The study provides new insights into the role of cell-cell interactions in regulating both cell differentiation and cell function and highlights the importance of developing appropriate differentiation protocols for tissue engineering therapies.

Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry.

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

[Determination of collagen in tendon by reversed-phase high performance liquid chromatography].

A method for determining collagen in tendon by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. After hydrolysis with hydrochloric acid, the collagen in samples was decomposed into hydroxyproline which hydroxyproline can be derivatized with 2, 4-dinitrochlorobenzene for the determination by HPLC (reversed-phase C18 column, 0.01 mol/L NaAc-HAc (pH 6.0)-CH3CN (80: 20, v/v) as mobile phase, detection at 360 nm). The factors influencing hydrolysis, derivatization and HPLC analysis were studied and optimized. Sixty samples were analyzed with the proposed method. The linear range was from 3 microg/L to 100 mg/L and the detection limit was 3 microg/L. The relative standard deviation (RSD) of determination was 1.95%. The recoveries of spiked samples were 98.4%-110.8%. The results show that the method is sensitive, accurate and suitable for tendon determination.

[Progress in vitreous cryopreservation of living bioproducts].

In this brief review, some key issues related to vitreous cryopreservation of living tissues (natural or engineered), including cells, embryos, tissues, organs, and engineered tissues, are outlined. The principle of vitreous cryopreservation for the biological activity and functionality is demonstrated. The procedures of cooling/ rewarming, composition and function of optimal cryoprotectants, and their effects on bioproducts are described. Vitrification could, therefore, prove to be a useful and effective method of bioproduct cryopreservation for a long period of time, particularly for organized tissues and organs. However, the toxicity of the cryoprotective agents and the devitrification occurring during the rewarming process need additional investigations. Several key areas of research on vitrification are also addressed.

Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis.

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.

[Characterization of insecticidal crystal proteins genes from Bacillus thuringiensis 4.0718 strain].

In this study, we rapidly identified Bacillus thuringiensis 4.0718 strain that harbored the known cryl and cry2 type genes by a PCR strategy. Three pairs of universal oligonucleotide primers were designed to detect all known cry1, cry2 and cry3 type gene sequences. Then the DNA of the positive strain 4.0718 was probed with a set of specific primers. One feature of this screening method was that each gene was expected to produce a PCR product having a precise molecular weight. PCR products having different sizes probably represented the gene was a potentially novel gene. Differentiations among these genes was determined on the basis of the electrophoresis patterns of PCR products. Finally, five cry1 type genes (cry1 Aa, cry1 Ab, cry1 Ac, cry1 Cb, a novel cry4.5 type genes) and one cry2 Ac type gene had been detected from Bacillus thuringiensis 4.0718 strain.