The dyeing effect of acridine orange for multiple plasmid systems is sensitive to temperature.

The Goldview dyeing of the natural multiplasmid system of Lactobacillus plantarum PC518 was affected by temperature. The article want to identify the specific molecules that cause temperature sensitivity, then experiment on the universality of temperature sensitivity, and finally preliminarily analyze the influencing factors. At 5°C and 25°C, single pDNA, multiplasmid system, and linear DNA samples were electrophoretic on agarose gel prestained by Goldview 1, 2, 3, and acridine orange (AO), respectively. Eighteen vectors of Escherichia coli and two vectors shortened by cloning were mixed into multiplasmid systems with different member numbers, and then electrophoresis with AO staining was performed within the range of 5°C-45°C, with a linearized multiplasmid system as the control. The lane profiles (peaks) were captured with Image Lab 5.1 software. After electrophoresis, the nine-plasmid-2 system was dyed with AO solutions of different ionic strengths to detect the effect of ionic strength on temperature sensitivity. It was measured that the UV-visible absorption spectra of the nine-plasmid-2 system dissolved in AO solutions with different ionic strengths and pH. Further, a response surface model was constructed using Design-Expert.V8.0.6 software. The electrophoresis result showed that the multiplasmid system from L. plantarum PC518 stained by AO staining showed a weak band at 5°C and five bands at 25°C, which was similar to the result of staining with Goldview 1, 2, and 3. The synthetic nine-plasmid-1 system and nine-plasmid-2 system displayed different band numbers on the electrophoresis gel in the electrophoresis temperature range of 5°C-45°C, namely 3, 4, 6, 4, and 2 bands, as well as 2, 6, 7, 8, and 5 bands. Using the 1× Tris-acetate-EDTA (TAE)-AO solution, the poststaining results of the nine-plasmid-2 system in the temperature range of 5°C-45°C were 4, 6, 9, 9, and 7 bands, respectively. Further, using 5×, 10×, or 25× TAE buffer, the AO poststaining results at 5°C were 4, 2, and 1 bands, respectively. The ultraviolet spectral results from 5°C to 25°C showed that there was a significant difference (3.5 times) in the fluctuation amplitude at the absorption peak of 261.2 nm between 0× and 1-10× TAE-AO solution containing the nine-plasmid-2 system. Specifically, the fluctuation amplitudes of 0×, 1×, 5×, and 10× samples were 0.032, 0.109, 0.112, and 0.110, respectively. At the same time, using 1× and 10× TAE buffer, the AO-stained linear nine-plasmid-2 system remained stable and did not display temperature sensitivity. The response surface models of the AO-stained nine-plasmid-2 system intuitively displayed that the absorbance of the 1× TAE samples increased significantly with increasing temperature compared to the 0× TAE samples, regardless of the pH value. The findings confirmed a temperature-dependent effect in AO staining of natural or synthetic multiplasmid systems, with the optimum staining result occurring at 25°C. Ion strength was a necessary condition for the temperature sensitivity mechanism. This study layed the groundwork for further investigation into the reasons or underlying mechanisms of temperature sensitivity in AO staining of multiplasmid systems.

Multiplasmid DNA in agarose gel electrophoresis: Suitable dye and temperature is essential for prominent profiles.

Nucleic acids dye Goldview is widely used in agarose gel electrophoresis (AGE). However, in this study, a sample of multiplasmid DNA (multi-pDNA) stained with Goldview analyzed by AGE showed its instability at low temperature. Three types of DNA samples were analyzed, including linear DNA (ladder), single-plasmid DNA (single-pDNA), and multi-pDNA, electrophoretic conditions were optimized by adjusting the dye, the buffer, and the temperature (1-50°C). The results showed that the light intensity of Gelred is 2.2-times higher than that of Goldview in staining multi-pDNA. Compared with the single-pDNA and the linear DNA, the multi-pDNA stained with Goldview was greatly affected by temperature. This short communication indicated that Gelred is a highly applicable dye for analyzing multiplasmid samples. The degree and the way of binding of Goldview to multi-pDNA are greatly affected by temperature.

Lactobacillus futsaii subsp. chongqingii subsp. nov., Isolated from a Traditional Chinese Pickle.

Strain CQ16Z1T was isolated from jamiecosley, a traditional Chinese pickle. The isolate was Gram-positive, non-motile, non-spore-forming, facultatively anaerobic, catalase-negative, and long rod-shaped. The optimal temperature for growth was 37 °C and the DNA G + C content was 39.1 mol%. The results of 16S rRNA and rpoA gene sequencing, DNA-DNA hybridization, and peptidoglycan type analyses indicated that strain CQ16Z1T belongs to the recognized species Lactobacillus futsaii. However, the analysis results of pheS gene sequencing, amplified fragment length polymorphism, phenotypic profiles, cellular fatty acid, cell-wall monosaccharide determination, and cell morphology revealed that the novel strain was obviously different from the type strain L. futsaii JCM17355T, and had genetic relationship with Weissella cibaria to a certain degree, suggesting that the novel strain represents a novel subspecies of L. futsaii, for which the following names are proposed: L. futsaii subsp. futsaii subsp. nov. (type strain YM0097T = JCM 17355T = BCRC 80278T) and L. futsaii subsp. chongqingii subsp.nov., with the type strain CQ16Z1T (= CCTCC AB2017187T = KCTC 21089T).

[Apoptosis of A549 cells induced by cloned Noxa gene].

OBJECTIVE: To clone the Noxa gene and to observe the apoptosis of A549 cells transfected with the recombinant plasmid of pcDNA-Noxa. METHODS: The Noxa gene was obtained by PCR, and was cloned into pcDNA3. 1(-). A549 cells were transfected with the recombinant plasmid of pcDNA-Noxa. Western blot analysis was performed to determine the overexpression of Noxa. A549 cells were stained with Hoechst 33258 to observe the apoptosis. RESULTS: The recombinant plasmid of pcDNA-Noxa was successfully constructed evidenced by endonuclease digestion and sequence analysis. The overexpression of Noxa was identified using Western blot analysis. The recombinant plasmid of pcDNA-Noxa induced apoptosis of A549 cells. CONCLUSION: Nora has exhibited potential pro-apoptotic activity against A549 cells. This study is a foundation for further research into pro-apoptotic activity of Noxa gene.

Whole genome sequence analysis of two subspecies of Companilactobacillus Futsaii and experimental verification of drug resistance and effect on the exploratory behavior of mice based on unique gene.

This study characterized the whole genome of Companilactobacillus futsaii subsp. chongqingii CQ16Z1 isolated from Chongqing of China, performed genome sequence analysis with Companilactobacillus futsaii subsp. futsaii YM0097 isolated from Taiwan of China, and experimentally verified drug resistance and effect on the exploratory behavior of male C57BL/6 mice and analysis of gut microbiota and metabolomic studies. The genome of CQ16Z1 is 2.6 Mb. Sequence analysis between genomes showed that the two strains are Companilactobacillus futsaii. The unique genes of CQ16Z1 and YM0097 are 217 and 267, which account for 9% and 11% of the whole genomes, respectively. According to unique gene annotation, the results showed that genes associated with carbohydrate metabolism, environmental information processing, metabolism of cofactors and vitamins, cell wall/membrane/envelope biogenesis, phage and drug resistance are significantly different. The results of the drug resistance experiment showed that YM0097 had different degrees of resistance to 13 antibiotics, while CQ16Z1 was sensitive to more than half of them. YM0097 contains 9 prophage regions and CQ16Z1 contains 3 prophage regions. The results of the open field test showed that the time (P = 0.005; P = 0.047) and distance (P < 0.010; P = 0.046) of the central area of Y97 group and CQ group are significantly different from the control group. The results of the elevated plus maze test showed that compared with the control group, Y97 group had significant differences in the number of entries to the open arms and the percentage of open arms entry times (P = 0.004; P = 0.025), while the difference between the CQ group and the control group was not significant. YM0097 has a more obvious effect on the exploratory behavior of mice. The effects of YM0097 and CQ16Z1 on the intestinal flora of mice are also different. YM0097 may be more beneficial to the intestinal flora of the host. And LC/MS also showed that the metabolic effects of the two strains on the host are different. Finally, we believe that YM0097 is more suitable for application research as a psychobiotics.

Hypericin blocks the function of HSV-1 alkaline nuclease and suppresses viral replication.

ETHNOPHARMACOLOGICAL RELEVANCE: Hypericum perforatum L. has a long history in many countries of being used as a herbal medicine. It is also widely used in Chinese herbal medicine for the treatment of infections. Hypericin, a main component extracted from Hypericum perforatum L., has attracted the attention of many researchers for its remarkable antiviral, antitumor and antidepressant effects. AIM OF THE STUDY: To find plant molecules that inhibit the alkaline nuclease (AN) of herpes simplex virus type 1 (HSV-1) and suppress viral replication. MATERIALS AND METHODS: Bioinformatics methods were used to determine which compounds from a variety of natural compounds in our laboratory interact with AN. By this means we predicted that hypericin may interact with AN and suppress HSV-1 replication. Experiments were then carried out to verify whether hypericin inhibits the bioactivity of AN. The Pichia pastoris expression system was used to obtain recombinant AN. The exonuclease and endonuclease activity of AN treated with hypericin were tested by electrophoresis. Immunohistochemical staining of the HSV-1 nucleocapsids was used to find out whether hypericin inhibits the intracellular function of AN. Real-time PCR and western blotting analysis were performed to test viral gene expression and viral protein synthesis. The extent of viral replication inhibited by hypericin was determined by a plaque assay and a time of addition assay. RESULTS: Recombinant AN was obtained by Pichia pastoris expression system. The exonuclease and endonuclease activity of recombinant AN were inhibited by hypericin in the electrophoresis assay. Hypericin showed no inhibitory effect on BeyoZonase™ Super Nuclease or DNase I. T5 Exonuclease activity was inhibited partially by10 µM hypericin, and was completely suppressed by 50 µM hypericin. Hind Ⅲ was inhibited by hypericin at concentrations greater than 100 µM, but EcoR I, BamH I, and Sal I were not inhibited by hypericin. HSV-1 nucleocapsids gathered in the nucleus when the viruses were treated with hypericin. Plaque formation was significantly reduced by hypericin (EC50 against HSV-1 F is 2.59 ± 0.08 µM and EC50 against HSV-1 SM44 is 2.94 ± 0.10 µM). UL12, ICP27, ICP8, gD, and UL53 gene expression (P < 0.01, 4.0 µM hypericin treated group vs control group) and ICP4 (P < 0.05, 6.0 µM hypericin treated group vs control group), ICP8 and gD (P < 0.05, 2.0 µM hypericin treated group vs control group) protein synthesis were inhibited by hypericin. In the time of addition assay, HSV-1 was suppressed by hypericin in the early stages of viral replication. Hypericin exhibits potent virucidal activity against HSV-1 and inhibits the adsorption and penetration of HSV-1. CONCLUSION: Hypericin inhibits the bioactivity of AN and suppresses HSV-1 replication. The data revealed a novel mechanism of the antiherpetic effect of hypericin.

Characterization of pLP18, a novel cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle.

A cryptic plasmid of Lactobacillus plantarum PC518 isolated from Chinese pickle, designated pLP18, was sequenced and characterized. It is a 1806-bp circular molecule with a G+C content of 37.5%. Sequence analysis of pLP18 revealed three putative open reading frames (ORFs), in which ORF1 contained conserved motifs of pMV158-family Rep proteins and showed 60% similarity with the Rep protein of pPSC22, a member of rolling-circle replication (RCR) pMV158 family. The double strand origin (dso) of pMV158 family and the single strand origin A (ssoA) located upstream of the rep gene. The putative cop and rnaII genes were predicted to be regulatory genes controlling copy number of pLP18. The results of Southern hybridization suggested that pLP18 replicate via the RCR mechanism. Furthermore, the relative copy number of pLP18 was estimated to be about 24 copies per chromosome equivalent by quantitative PCR.

The therapeutic potential of attenuated diphtheria toxin delivered by an adenovirus vector with survivin promoter on human lung cancer cells.

Adenoviral vectors are superior to plasmid vectors in their gene transport efficiency. The A subunit of the diphtheria toxin (DTA) gene is a popular suicide gene in cancer gene therapy. However, DTA is seldom used in adenoviral therapy due to its great toxicity. The toxicity of DTA is so great that even a single molecule of DTA is enough to kill one cell. To avoid this highly toxic effect on normal cells, DTA should be controlled by tumor-specific promoters. The survivin promoter is a widely used tumor-specific promoter. But genes driven by the survivin promoter show a low level of basal gene expression in non-cancer cells. DTA driven by the survivin promoter in adenoviral vectors may be highly toxic not only to cancer cells but also to normal cells. Therefore, DTA should be attenuated when it is used in adenoviral vectors driven by the survivin promoter. In this study, we compared the three kinds of recombinant adenoviruses that carry DTA or its attenuated forms (DTA176 and DTA197) in the treatment of human lung cancer. The results showed that in comparison with both DTA and DTA176, DTA197 is more suitable for adenoviral cancer therapy controlled by the survivin promoter. In addition, Adsur-DTA197 (DTA197 delivered by an adenoviral vector with the survivin promoter) sensitized human lung cancer cells to cisplatin both in vitro and in vivo. These results indicated that Adsur-DTA197 may be a potential chemosensitizer in cancer therapy.

Functional identification of a putative beta-galactosidase gene in the special lac gene cluster of Lactobacillus acidophilus.

The putative beta-galactosidase gene (lacZ) of Lactobacillus acidophilus has a very low degree of homology to the Escherichia coli beta-galactosidase gene (lacZ) and locates in a special lac gene cluster which contains two beta-galactosidase genes. No functional characteristic of the putative beta-galactosidase has been described so far. In this study, the lacZ gene of L. acidophilus was hetero-expressed in E. coli and the recombinant protein was purified by a three-step procedure. The product of the lacZ gene was also extracted from L. acidophilus ATCC 4356 and active staining was carried out. The enzymatic properties of the purified recombinant LacZ were assayed. The results of hetero-expression showed the recombinant LacZ without tag had beta-galactosidase activity. The purified recombinant LacZ had a specific activity of 43.2 U/mg protein. The result of active staining showed that the functional product of the lacZ gene did exist in L. acidophilus. The L. acidophilus beta-galactosidase (LacZ) had an optimal pH of 6, an optimal temperature of 37 degrees C and could hydrolyze 73% of lactose in milk in 30 h at 10 degrees C. The L. acidophilus beta-galactosidase (LacZ) was identified as cold-adapted beta-galactosidase in this study for the first time, and may be useful for lactose removal from dairy products at low temperatures.

Application of TonB-Dependent Transporters in Vaccine Development of Gram-Negative Bacteria.

Multiple scarce nutrients, such as iron and nickel, are essential for bacterial growth. Gram-negative bacteria secrete chelators to bind these nutrients from the environment competitively. The transport of the resulting complexes into bacterial cells is mediated by TonB-dependent transporters (TBDTs) located at the outer membrane in Gram-negative bacteria. The characteristics of TBDTs, including surface exposure, protective immunogenicity, wide distribution, inducible expression in vivo, and essential roles in pathogenicity, make them excellent candidates for vaccine development. The possible application of a large number of TBDTs in immune control of the corresponding pathogens has been recently investigated. This paper summarizes the latest progresses and current major issues in the application.

Identification and experimental verification of protective antigens against Streptococcus suis serotype 2 based on genome sequence analysis.

Streptococcus suis serotype 2 (S. suis 2) is an important zoonotic pathogen that can cause severe disease and even death in both humans and swine. No effective vaccine is clinically available. In this study, a reverse vaccinology method was first applied to identify protective antigens against S. suis 2. As a consequence, 153 genes encoding vaccine candidates were selected from the whole genome sequence by means of bioinformatics analysis, from which 10 genes were selected based on experimental evidences arising from the study of related bacteria such as Streptococcus pneumoniae, group B streptococcus, S. suis and so on. Of 10 target genes, 8 were successfully expressed in Escherichia coli Rosetta, and expressed proteins were purified and used as the immunogens for evaluating vaccine efficacy in a mouse infection model. The results have confirmed that RTX family exoprotein A (RfeA), epidermal surface antigen (ESA), immunoglobulin G (IgG)-binding protein (IBP), and suilysin (SLY) can induce a protective response of the vaccinated animals against S. suis 2, whereas RfeA, ESA, and IBP mainly induce humoral-mediated immunity, and SLY elicits a combined pattern of both humoral- and cellular-mediated immunity. Although immunoprotection of SLY against S. suis 2 was reported previously, RfeA, ESA, and IBP were explored first in this study.

Detection and characterization of a theta-replicating plasmid pLP60 from Lactobacillus plantarum PC518 by inverse PCR.

Plasmid DNA of Lactobacillus plantarum PC518 was isolated by an improved method which contained a washing step for removing lysozyme. Three plasmid DNA libraries were constructed. A pair of outward primers was designed at both ends of the novel plasmid fragment obtained from plasmid DNA libraries, and the remainder of the circle plasmid was amplified by inverse PCR (iPCR). The whole sequence of plasmid was analyzed by the basic local alignment search tool, Tandem Repeats Finder, DNAMAN V6.0, DNASTAR and MEGA X software. The copy number was measured using quantitative real-time PCR. Plasmid extract showed 7 bands on agarose gel, indicating that L. plantarum PC518 contains multiple plasmids. The complete sequence of plasmid pLP60 was obtained by plasmid DNA libraries and iPCR. pLP60 is 6006 bp in length with a G + C content of 41.19 %, which encodes 8 open reading frames (ORFs). The ori site like theta-type could be located upstream of repB, which contains a short tandem repeats (sTR) and a long tandem repeats (lTR). RepB of pLP60 only had low similarity with Rep protein of known theta-type plasmids, but phylogenetic tree analysis showed that plasmids whose Rep proteins are similar to pLP60 have lTR at ori, and the conservativeness of lTR is consistent with similarity of Rep proteins, suggesting that RepB of pLP60 is a theta-replicating protein. So pLP60 was classified as class A of theta replication. The copy number of pLP60 was measured as 5 copies per cell by qPCR.

Genome Sequence of Lactobacillus futsaii Y97, a Potential Probiotic Strain Isolated from Futsai of Taiwan.

Here, we report the complete genome sequence of Lactobacillus futsaii Y97, a potential probiotic strain isolated from futsai of Taiwan. The genome consists of one chromosome of 2.56 Mb and three plasmids. The genome contains 2,622 genes, which make up 87.06% of the genome.

[Cloning, expression and characterization of a heterodimeric beta-galactosidase from Lactobacillus acidophilus ATCC 4356].

UNLABELLED: Heterodimeric beta-galactosidase of Lactobacillus acidophilus belongs to glycoside hydrolase family 2, encoded by two overlapping and translational coupling genes, lacL and lacM. The lacL and lacM genes of the sequenced strain L. acidophilus NCFM encode polypeptides with calculated molecular masses of 73,253 and 35,817 Da, respectively. OBJECTIVE: To clone, overexpress and characterize the enzyme in Escherichia coli. METHODS: We cloned the fragment (2834 bp) containing ribose-binding site (RBS) and coding regions of the lacLM genes from L. acidophilus ATCC 4356 into expression vector pQE31. RBS and HIS-Tag of pQE31 were substituted by inserted fragment. Recombinant plasmid was electrotransformed into E. coli JM109. Expression product was purified by ammonium sulphate fractionation, anion-exchange, affinity chromatography and gel permeation. Native molecular mass of homogenous enzyme was measured by gel permeation, and beta-galactosidase activity was determined by using o-nitrophenyl-beta-D-galactopyranoside (ONPG) as the substrate. RESULTS: Overexpression of the soluble enzyme in E. coli was achieved. Amino acid residue 512 of recombinant LacL was different from that of L. acidophilus NCFM. Homogenous enzyme was obtained by purification. The homogenous enzyme had a specific activity of 226 U/mg protein, a native molecular mass of 96.3+/-4.6 kDa, an optimum temperature at 49 degrees C and an optimum pH of 7. The Km and Vmax values of the enzyme were 2.18+/-0.12 mmol/L, 273+/-5 U/mg protein respectively.

Genome Sequence of Weissella cibaria M2, a Potential Probiotic Strain Isolated from the Feces of a Giant Panda.

Here, we report the complete genome sequence of Weissella cibaria M2, a potential probiotic strain isolated from the feces of a giant panda (Ailuropoda melanoleuca). The genome consists of one chromosome of 2.56 Mb and two plasmids. The genome contains 2,420 genes which make up 86.17% of genome.

p53 Plays a Key Role in the Apoptosis of Human Ovarian Cancer Cells Induced by Adenovirus-Mediated CRM197.

Cross-reacting material 197 (CRM197) is a mutant form of the diphtheria toxin. Recent studies have found that CRM197 exerts an experimental antitumor effect on several types of tumors. This study applied a novel treatment of adenovirus-mediated CRM197 (AdCRM197) to human ovarian cancer cells. Interestingly, it was found that A2780 cells were sensitive to AdCRM197, but SKOV3 cells were resistant to it. Since SKOV3 cells are p53 deletion cells, while A2780 cells are p53 wild-type cells, it was postulated that p53 might play a key role in AdCRM197-induced apoptosis. This presumption was demonstrated by means of knockdown of p53 of the A2780 cells through lentivirus-mediated RNA interference. This knockdown resulted in the A2780 cells becoming resistant to AdCRM197. To verify this presumption further, the wild-type p53 gene in the SKOV3 cells was replaced with adenovirus-mediated p53 (Adp53). As expected, AdCRM197 plus Adp53 resulted in apoptosis of the SKOV3 cells. The combined treatment of AdCRM197 plus Adp53 also showed a good antitumor effect in the in vivo experiment on nude mice with xenograft tumors. Taking these results together, it is concluded that AdCRM197 induces apoptosis of human ovarian cancer cells via the p53 pathway. Moreover, it was found that Adp53 can reverse the resistance of p53-deletion human ovarian cancer cells to AdCRM197. The combination of AdCRM197 and Adp53 may be a potentially effective method for overcoming the resistance of p53-deficient human ovarian cancer to AdCRM197.

Anti-angiogenic genistein inhibits VEGF-induced endothelial cell activation by decreasing PTK activity and MAPK activation.

Genistein (Gen), a soy isoflavone, is considered to exert potent antitumor effect partially through its anti-angiogenesis property. However, the precise molecular mechanism is still unknown. Our previous investigations have demonstrated that genistein down-regulates expression of pro-angiogenic factors via inhibiting protein tyrosine kinase (PTK) activity both in breast cancer cells and in xenograft tumors. In the present experiment, we chose cultured human umbilical vein endothelial cells (HUVECs), which have a considerable role in tumor angiogenesis formation, to explore the influence of genistein on VEGF-stimulated endothelial cell activation and the underlying mechanism. Stimulation of human primary HUVECs by VEGF not only increased endothelial cell protein tyrosine kinase (PTK) activity but also augmented matrix metalloproteinase-2 (MMP-2), -9 secretions and increased MMP-2, -9 activities. Treatment of ECs with genistein induced VEGF-loaded endothelial apoptosis by inhibiting production and activity of matrix metalloproteinases (MMPs). In addition, exposure to genistein decreased activation of JNK and p38, not ERK-1/2, induced by VEGF. Collectively, our findings suggested that the inhibition of PTK activity and MAPK activation and the decrease in MMPs production and activity by genistein interrupt VEGF-stimulated endothelial cell activation, which thereby may represent a mechanism that would explain the anti-angiogenesis effect of genistein and its cancer-protective function.

[Study on expression of Streptococcus suis serotype 2 sly gene, purification and activity of its product].

OBJECTIVE: To clone and express Streptococcus suis serotype 2 (S. suis 2) sly gene for constructing an foundation on identification of S. suis 2 protective antigen. METHODS: The sly gene was amplified from S. suis 2 clinical isolate strain 05ZYH33 genome DNA by PCR. The gene fragment was inserted into the expression vector pET-30b(+) to build pET30b-sly. When recombinant vector pET30b-sly was identified by restriction enzyme cutting and DNA sequencing as a correct one, subsequently it was transformed to E. coli Rosetta for expression under IPTG induction. The obtained fusion protein was purified by Ni-NTA affinity chromatography. The immunologic and hemolysis activity of the purified protein was proved through Western blot and hemolysis assay respectively. RESULTS: The PCR product was around 1500 bp. The gene segment inserted into the recombinant vector was proven to be completely identical with the sly gene sequence in the total genome sequence of S. suis 2. The target protein expressed was up to 30% of the total somatic protein under IPTG induction. The protein purity reached above 80% after purification. The protein could be recognized by human serum infected with S. suis 2 and could dissolve swine erythrocytes with the Hemolytic titer as 256. CONCLUSION: The expression vector pET30b-sly was successfully constructed. The target protein could be over-expressed in E. coli and possessed its biological activity after purification.