Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis / 生物工程学报

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPKK81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPKK81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

Serratia nevei 9rpt1, a Potential Microorganism for Phosphorus Recovery

Aims: Phosphorus (P) is an essential element for life, finite and irreplaceable. Its constant exploration on a global scale has motivated frequent alerts regarding an eventual crisis due to the shortage of this nutrient. However, it is possible to recycle it and reintroduce it into the ecological cycle. One viable alternative is the microbial recovery of phosphate. Study design: This study is based on systematic bioprospection of bacteria in phosphate-deficient Amazon regions. Place and Duration of Study: Bacteria were isolated from black water samples, collected in the Rio Pretinho, located at Serra do Aracá, Barcelos, Amazonas, Brazil, from January to July 2019. Methodology: Microbial isolation was performed in Luria Bertani agar medium. For the genomic study, the isolate with the best performance in the phosphate uptake test was chosen. The WGS was carried out in a Illumina HiSeq 2500 System. The assembly of the draft genome was carried out with the SPAdes and the annotation by the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Results: Serratia nevei 9rpt1 recovers 90% of the phosphate available in the culture medium. Its draft genome comprises 5.4 MB, the GC content is 59.52% and 4,922 coding sequences were identified, among these, two pst operons: one complete, containing the five pst genes and one missing pstS, pstC and phoU genes. Conclusion: Serratia nevei 9rpt1, isolated from an Amazonian environment poor in phosphate, is very efficient to uptake this nutrient in a Pi starvation condition. The genomic findings revealed this strain has an additional high affinity Pi uptake pst system containing the ATP-binding protein PstB, the canonical permease PstA, a putative permease other than PstC, upstream of the PstA and two essential enzymes in the polyphosphate metabolism: polyphosphate kinase 1 and exopolyphosphatase.

Expression of polyphosphate kinase from Sphingobacterium siyangensis and its application in ATP regeneration system / 生物工程学报

Polyphosphate kinase plays an important role in the catalytic synthesis of ATP in vitro. In order to find a polyphosphate kinase that can efficiently synthesize ATP using short-chain polyphosphate (polyP) as substrate, the polyphosphate kinase 2 (PPK2) from Sphingobacterium siyangensis was cloned and expressed in Escherichia coli BL21(DE3). As an enzyme for ATP regeneration, PPK2 was used in combination with l-amino acid ligase (YwfE) to produce l-alanyl-l-glutamine (Ala-Gln). The length of ppk2 of S. siyangensis is 810 bp, encoding 270 amino acids. The SDS-PAGE showed that PPK2 was expressed correctly and its molecular weight was 29.7 kDa as expected. The reaction conditions of PPK2 were optimized. PPK2 could maintain good activity in the range of 22-42 ℃ and pH 7-10. The highest enzyme activity was observed at 37 ℃, pH 7, 30 mmol/L magnesium ion (Mg2+), 5 mmol/L ADP and 10 mmol/L sodium hexametaphosphate, and the yield of ATP reached 60% of the theoretical value in 0.5 hours at this condition. When used in combination with YwfE to produce Ala-Gln, the PPK2 showed a good applicability as an ATP regeneration system, and the effect was similar to that of direct addition of ATP. The PPK2 from S. siyangensis shows good performance in a wide range of temperature and pH, synthesizes ATP with cheap and readily available short chain polyP as substrate. The PPK2 thus provides a new enzyme source for ATP dependent catalytic reaction system.

Effects of microRNA-4286 on the growth, migration and invasion of gastric cancer cell line HGC-27 by regulating inositol polyphosphate-4-phosphatase type I / 解剖学报

Objective To investigate the effects of microRNA-4286 (miRNA-4286) on the growth, migration and invasion of gastric cancer cell line HGC-27 by regulating inositol polyphosphate-4-phosphatase type I (INPP4A). Methods HGC-27 cells were divided into blank control group, negative control group and intervention group. The blank control group received no treatment, negative control group was transfected with pEGFP-Nl plasmid, and intervention group was transfected with pEGFP-Nl-miRNA-4286 plasmid. Real-time PCR was used to detect the expression of miRNA-4286 in HGC-27 cells, and Western blotting was used to detect the expression of INPP4A protein in HGC-27 cells. The proliferation rate, invasion ability and migration ability of HGC-27 cells were detected by cholecystokinin octapeptide, cholecystokinin octapeptide (CCK8), Transwell chamber assay and scratch test, respectively. Results The miRNA-4286 expression level, proliferation rate, and number of transmembrane cells in intervention group were significantly higher than those in blank control group and negative control group (P0.05). Conclusion miRNA-4286 may promote the growth, migration and invasion of gastric cancer cell line HGC-27 by down- regulating the expression of INPP4A.

Concomitant use of immobilized uridine-cytidine kinase and polyphosphate kinase for 5'-cytidine monophosphate production / 生物工程学报

Uridine-cytidine kinase, an important catalyst in the compensation pathway of nucleotide metabolism, can catalyze the phosphorylation reaction of cytidine to 5'-cytidine monophosphate (CMP), but the reaction needs NTP as the phosphate donor. To increase the production efficiency of CMP, uridine-cytidine kinase gene from Thermus thermophilus HB8 and polyphosphate kinase gene from Rhodobacter sphaeroides were cloned and expressed in Escherichia coli BL21(DE3). Uridine-cytidine kinase was used for the generation of CMP from cytidine and ATP, and polyphosphate kinase was used for the regeneration of ATP. Then, the D403 metal chelate resin was used to adsorb Ni²⁺ to form an immobilized carrier, and the immobilized carrier was specifically combined with the recombinant enzymes to form the immobilized enzymes. Finally, single-factor optimization experiment was carried out to determine the reaction conditions of the immobilized enzyme. At 30 °C and pH 8.0, 60 mmol/L cytidine and 0.5 mmol/L ATP were used as substrates to achieve 5 batches of high-efficiency continuous catalytic reaction, and the average molar yield of CMP reached 91.2%. The above method has the advantages of low reaction cost, high product yield and high enzyme utilization rate, and has good applied value for industrial production.

Preparation of polypyrrole/nanosilica composite for solid-phase microextraction of bisphenol and phthalates migrated from containers to eye drops and injection solutions / 药物分析学报

This paper describes the electrodeposition of polyphosphate-doped polypyrrole/nanosilica nano-composite coating on steel wire for direct solid-phase microextraction of bisphenol A and five phthalates. We optimized influencing parameters on the extraction efficiency and morphology of the nanocomposite such as deposition potential, concentration of pyrrole and polyphosphate, deposition time and the nanosilica amount. Under the optimized conditions, characterization of the nanocomposite was inves-tigated by scanning electron microscopy and Fourier transform infra-red spectroscopy. Also, the factors related to the solid-phase microextraction method including desorption temperature and time, extrac-tion temperature and time, ionic strength and pH were studied in detail. Subsequently, the proposed method was validated by gas chromatography-mass spectrometry by thermal desorption and acceptable figures of merit were obtained. The linearity of the calibration curves was between 0.01 and 50 ng/mL with acceptable correlation coefficients (0.9956-0.9987) and limits of detection were in the range 0.002-0.01 ng/mL. Relative standard deviations in terms of intra-day and inter-day by five replicate analyses from aqueous solutions containing 0.1 ng/mL of target analytes were in the range 3.3％-5.4％ and 5％-7.1％, respectively. Fiber-to-fiber reproducibilities were measured for three different fibers prepared in the same conditions and the results were between 7.3％ and 9.8％. Also, extraction recoveries at two different concentrations were ≥96％. Finally, the suitability of the proposed method was demonstrated through its application to the analysis of some eye drops and injection solutions.

Screening of lung cancer tumor-associated suppressor genes using shRNA library targeting human transcriptome / 实用肿瘤学杂志

Objective The shRNA library was used to screen the tumor suppressor genes related to lung epithelial cells,and its function of inhibiting malignant transformation in lung cells was preliminarily verified,which provided a theoretical basis and a new therapeutic target for tumor prevention and treatment. Methods The shRNA retrovirus library was constructed by GP2 -293 virus packaging cell line to infect the immortalized lung bronchial epithelial BEAS-2B cells. The transfected epithelial cell clones were screened by soft agar colony formation,and a single transformed cell clone with a diameter greater than 1. 0 mm was selected. The in-serted shRNA fragment was amplified by PCR,and the target candidate gene corresponding to shRNA was determined by the conven-tional DNA sequencing and blast alignment. A candidate tumor suppressor gene INPP4B was verified by soft agar cloning and tumor formation in nude mice. MTT assay was used to detect the cell proliferation. Results Six lung epithelial malignant transformation in-hibitory factors were screened by soft agar colony formation. The candidate INPP4B gene was selected for functional experiments. Si-lencing INPP4B gene in BEAS-2B cells promoted the formation of clones in the soft agar plates,and the cell proliferation rate was accelerated. The silencing cell line showed the enhanced tumorigenicity in nude mice,indicating that INPP4B was involved in tumor formation. Conclusion shRNA library and soft agar colony formation assays are a powerful tool for screening tumor suppressor genes, and INPP4B is a malignant transformation inhibitor of lung epithelial cells.

Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

Objective·To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro.Methods·The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract,and the PPK2 phylogenetic tree was constructed.The binding affinity of the PPK2 aptamer to H37Rv,BCG,Mycobacterium smegmatis,Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA).The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis.The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method.H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed.H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d,absorbance at 600 nm was measured by microplate reader.The effect of PPK2 aptamer on the growth of H37Rv was observed.Results·PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract,and it was relatively close to Pseudomonas aeruginosa.The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv.Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h.The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L.The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth.Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration,which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth.Conclusion·PPK2 aptamer has good antibacterial activity against H37Rv in vitro.

Evaluation of anti-tuberculosis effect of Mycobacterium tuberculosis polyphosphate kinase 2 aptamer / 上海交通大学学报（医学版）

Objective: To investigate the bacteriostasis effect of Mycobacterium tuberculosis (MTB) polyphosphate kinase 2 (PPK2) aptamer on MTB in vitro. Methods: The bioinformatics method was used to analyze the homology of MTB PPK2 and common pathogens of respiratory tract, and the PPK2 phylogenetic tree was constructed. The binding affinity of the PPK2 aptamer to H37Rv, BCG, Mycobacterium smegmatis, Pseudomonas aeruginosa and Acinetobacter baumannii was analyzed by enzyme-linked oligonucleotide assay (ELONA). The PPK2 aptamer was incubated for 24 h in serum and its biological stability in serum was analyzed by agarose gel electrophoresis. The minimum inhibitory concentration (MIC) of the PPK2 aptamer to H37Rv was determined by micro-azure method. H37Rv was inoculated with 1 μmol/L PPK2 aptamer or random sequence on Roche culture medium for 10 d and colony growth status was observed. H37Rv was co-cultured with different concentrations of PPK2 aptamer for 10 d, absorbance at 600 nm was measured by microplate reader. The effect of PPK2 aptamer on the growth of H37Rv was observed. Results: PPK2 phylogenetic tree constructed by bioinformatics analysis showed that PPK2 protein of H37Rv was not closely related to the common pathogens of respiratory tract, and it was relatively close to Pseudomonas aeruginosa. The ELONA assay results showed that the PPK2 aptamer binded selectively to H37Rv. Agarose gel electrophoresis analysis showed PPK2 aptamer in serum was at least stable for 8 h. The MIC of the PPK2 aptamer to H37Rv was 50 nmol/L. The colony growth of Roche culture showed that PPK2 aptamer had an inhibitory effect on H37Rv growth. Growth inhibition test showed that the absorbance at 600 nm of H37Rv showed a decreasing trend with the increase of PPK2 aptamer concentration, which indicated that PPK2 aptamer had an inhibitory effect on H37Rv growth. Conclusion: PPK2 aptamer has good antibacterial activity against H37Rv in vitro.

Polyphosphate kinase 1-coding gene (ppk1) is involved in the oxidative stress resistance in uropathogenic Escherichia coli by modulating the expression of katG and katE genes / 中华微生物学和免疫学杂志

Objective To investigate the role and the mechanism of ppk1 gene (coding for polyphosphate kinase 1) in oxidative stress resistance in uropathogenic Escherichia coli (UPEC).MethodsMutant strains with ppk1-deletion (△pk1) and complemented strains (△pk1-C) were constructed based on the UPEC strain CFT073.A comparative analysis was conducted to analyze survival rates of CFT073, △pk1 and △pk1-C strains at different time points while they were under oxidative stress.Differences in protein expression between CFT073 and △pk1 strains were analyzed using mass spectrometric analysis.Differences between CFT073 and △pk1 strains in expression of katG and katE genes were analyzed using real-time quantitative RT-PCR.Results The survival rate of △pk1 strains was lower than that of CFT073 strains at every time point, while the survival rate of △pk1-C strains was basically the same as that of CFT073 strains.Gel image analysis and mass spectrometric analysis revealed that six proteins were down-regulated and one was up-regulated in △pk1 strains as compared with those in CFT073 strains.Expression of the catalase-coding genes katG and katE in △pk1 strains were respectively (20.5±8.2)% and (20.9±6.9)% of those in CFT073 strains (P<0.05).Conclusion The ppk1 gene plays an important role in oxidative stress resistance in UPEC by modulating the expression of catalase-coding genes katG and katE.

Construction of recombinant strains co-expressing PPK and GMAS for the synthesis of L-theanine / 生物工程学报

Recombinant strains expressing enzymes for ATP regeneration and L-theanine production were constructed and used for the synthesis of L-theanine. The ppk gene encoding polyphosphate kinase (PPK) from Rhodobacter sphaeroides and gmas gene encoding γ-glutamylmethylamide synthetase (GMAS) from Methylovorus mays were synthesized, and two recombinant plasmids, pETDuet-ppk+gmas and pET21a-ppk+gmas were constructed for co-expression of PPK and GMAS in Escherichia coli BL21(DE3). SDS-PAGE analysis showed that PPK and GMAS were overexpressed in soluble form in both recombinant strains. GMAS-PPK obtained from the recombinant strain containing pET21a-ppk+gmas was more efficient to synthesize L-theanine. After 24 h at 37 ℃ and pH at 7.0, 86.0% yield of L-theanine was achieved with catalytic amount of ATP. This study extends the application of enzymatic ATP regeneration system. In addition, it provides an efficient method for the biosynthesis of L-theanine.

Copper-induced adaptation, oxidative stress and its tolerance in Aspergillus niger UCP1261

Background The effects of exposure to copper, during growth, on the production of biomass, total protein, catalase, glutathione-S transferase, glutathione peroxidase, peroxidase, polyphosphate, acid and alkaline phosphatases, ultrastructure and the ability to remove this metal from Aspergillus niger, obtained from caatinga soil, were evaluated. Results All parameters tested were influenced by the concentration of metal in the culture medium. The presence of metal induced high levels of antioxidant enzymes, including lipid peroxidation, thereby revealing the appearance of an oxidative stress response. The variation in polyphosphate levels indicates the participation of the polymer in response to stress induced by copper. The activities of the phosphatases were positively influenced by growing them in the presence of copper. Ultrastructure changes in the cell surface, electron density, thickness, and septation were visualized by exposing cells to increasingly larger concentrations of metal. The isolate was able to remove the agent from the growth medium, while maintaining its physiological functions. The metal removed from the cultures exposed to 0.5 mM, 1 mM and 2 mM copper exhibited percentages of removal equivalent to 75.78%, 66.04% and 33.51%. Conclusions The results indicate that the isolate was able to grow in high concentrations of copper, activates mechanisms for adaptation and tolerance in the presence of metal, and is highly efficient at removing the agent. Such data are fundamental if a better understanding is to be reached of the cellular and molecular abilities of native isolates, which can be used to develop bioprocesses in environmental and industrial areas.

Multiple antibiotic susceptibility of polyphosphate kinase mutants (ppk1 and ppk2) from Pseudomonas aeruginosa PAO1 as revealed by global phenotypic analysis

BACKGROUND: Pseudomonas aeruginosa is known to be a multidrug resistant opportunistic pathogen. Particularly, P. aeruginosa PAO1 polyphosphate kinase mutant (ppk1) is deficient in motility, quorum sensing, biofilm formation and virulence. FINDINGS: By using Phenotypic Microarrays (PM) we analyzed near 2000 phenotypes of P. aeruginosa PAO1 polyP kinase mutants (ppk1 and ppk2). We found that both ppk mutants shared most of the phenotypic changes and interestingly many of them related to susceptibility toward numerous and different type of antibiotics such as Ciprofloxacin, Chloramphenicol and Rifampicin. CONCLUSIONS: Combining the fact that ppk1 mutants have reduced virulence and are more susceptible to antibiotics, polyP synthesis and particularly PPK1, is a good target for the design of molecules with anti-virulence and anti-persistence properties.

Concentração de flúor e cálcio no fluido do biofilme associada ao uso de dentifrícios fluoretados suplementados com trimetafosfato de sódio ou glicerofosfato de cálcio, sob desafio cariogênico / Fluoride and calcium concentration in the biofilm fluid associated with fluoridated dentifrices supplemented with sodium trimetaphosphate or calcium glycerophosphate, under cariogenic challenge

Estudos recentes demonstraram que dentifrícios com concentração reduzida de fluoreto (DCRF, 550 μg F/g) suplementados com cálcio ou fosfato apresentam efetividade clínica semelhante à de um dentifrício convencional (DC, 1100 μg F/g). Entretanto, o mecanismo pelo qual estes compostos atuam nos processos de des- e remineralização ainda é incerto. O presente estudo avaliou a concentração de F e Ca no fluido do biofilme formado in situ sob desafio cariogênico após o uso de dentifrícios fluoretados, suplementados ou não com trimetafosfato de sódio (TMP) ou glicerofosfato de cálcio (CaGP). Voluntários (n=12) foram aleatoriamente divididos em 5 grupos, de acordo com os seguintes dentifrícios: Placebo (sem F, TMP ou CaGP), DC, DCRF sem suplementação (550F) e DCRF suplementado com 1% TMP (550F-TMP) ou 0,25% CaGP (550F-CaGP). Em cada fase, os voluntários utilizaram um dispositivo palatino contendo 4 blocos de esmalte bovino. O desafio cariogênico foi realizado com solução de sacarose 30%, 6 vezes ao dia. Na manhã do 8º dia, o biofilme foi coletado 1h e 12h após a escovação e desafio cariogênico. As análises de F e Ca foram realizadas com eletrodo invertido após tamponamento com TISAB III e por espectrofotometria (Arsenazo III), respectivamente. Os dados foram submetidos a ANOVA a 2 critérios (medidas repetidas) e teste de Student-Newman-Keuls (p<0,05). Uma relação dose-resposta entre as concentrações de F nos dentifrícios e no fluido do biofilme foi verificada...

Recent studies demonstrated that low fluoride dentifrices (LFD, 550 μg F/g) supplemented with calcium or phosphate have a similar effectiveness to a conventional dentifrice (CD, 1100 μg F/g). However, the mechanisms by which these compounds act on the de- and remineralization processes remain unclear. The present study evaluated fluoride (F) and calcium (Ca) concentrations in the biofilm fluid formed in situ under cariogenic challenge after using F dentifrices, supplemented or not with sodium trimetaphosphate (TMP) or calcium glycerophosphate (CaGP). Volunteers (n=12) were randomly divided into 5 groups according to the following toothpastes: Placebo (no F or CaGP, TMP), CD and LFD with no supplementation (550F) or supplemented with 1% TMP (550F-TMP) or 0.25% CaGP (550F-CaGP). In each phase, volunteers wore palatal appliances containing 4 bovine enamel blocks. The cariogenic challenge was produced using a 30% sucrose solution, 6 times a day. On the morning of the 8th day, biofilm samples were collected 1h and 12h after brushing and cariogenic challenge. F and Ca analyzes were performed with the inverted electrode after buffering with TISAB III and using the Arsenazo III method, respectively. Data were submitted to 2-way ANOVA (repeated measures) and Student-Newman-Keuls test (p<0.05). A dose-response relationship was verified between F concentrations in the dentifrices and those in the biofilm fluid. Significant differences were observed among Placebo, 550F and CD only 1 h after brushing, without statistical differences among 550F, 550F-TMP and 550F-CaGP. No defined trend was observed among the groups regarding Ca concentrations in the biofilm fluid, with the highest values found for Placebo and 500F-CaGP. It was concluded that the anticaries effects of LFDs supplemented with CaGP or TMP cannot be related to an increased availability of F and Ca in the biofilm fluid...

Construction of uropathogenic Escherichia coli strain with ppk1 gene deletion and study on its biolog-ical properties / 中华微生物学和免疫学杂志

Objective To construct a Polyphosphate kinase 1 ( ppk1) gene deletion mutant of uro-pathogenic Escherichia coli (E.coli) CFT073, and to explore the biological properties of the mutant strain . Methods The ppk1 gene deletion strain (△pk1) was constructed based on CFT073 E.coli strain by usingλRed homologous recombination technology .A comparison analysis was conducted on adhesive and invasive abilities between CFT073 wild type strain and △pk1 strain in in vitro model of human bladder cancer epithe-lial cell 5637 .Crystal violet staining method was used to evaluate the influences of ppk1 gene deletion on biofilm formation.Results The CFT073 ppk1 deletion mutant strain was successfully constructed .Com-pared with the wild type strain ,△pk1 strain showed impaired adhesive and invasive abilities to 5637 cells. Moreover , the absorbance values of crystal violet at 570 nm at each time point of the mutant strain group were also lower than those of the wild-type strain group .Conclusion The ppk1 gene deletion mutant of uro-pathogenic E.coli CFT073 could be successfully constructed by Red homologous recombination technology and its biological properties indicates that ppk1 gene plays an important role in the pathogenesis of uropatho-genic E.coli infection through regulating the abilities of adhesion , invasion and biofilm formation .

Development and evaluation of protective capacity of Salmonella Enteritidis polyphosphate kinase-deleted and temperature-sensitive mutant / 대한수의학회지

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.

The effect of polyphosphate on exophytic bone formation / 대한치주과학회지

PURPOSE: It has been shown that the inorganic polyphosphate is effective for the regeneration of bones through the preliminary animal test of rabbits. The most effective concentration of the polyphosphate, however, is not known yet. Moreover, the effectiveness of carriers inside human body is not confirmed.. MATERIALS AND METHODS: In this study, we examined the effect of the concentration of the inorganic polyphosphate on the process of the bone regeneration using the 6 weeks old rabbits with the weight of 2.0 kg in average. We performed the experiment using TR-ePTFE membrane(membrane) filled with collagen immersed in 4%, 8% of inorganic polyphosphate, respectively, following removal of the proper sized cortical bones from the rabbit calvaria. The experimental results were compared with the one of the following four groups: The negative control group for membrane only, the positive control group for membrane filled with collagen, the first experimental group for membrane filled with collagen immersed in 4% of inorganic polyphosphate, and the second experimental group for membrane filled with collagen immerse in 8% of inorganic polyphosphate. The fragments of the tissue with membrane obtained from each group of the sacrificed rabbits for 8 or 16 weeks sustained after surgery were then prestained by the Hematoxylin-Eosin stain and coated by resin to form non-decalcified specimens for the histologic examination and analysis. New bone formation was assessed by histomorphometric and statistical analysis. RESULTS: 1. All groups have shown better bone regeneration at 16weeks than 8weeks. 2. Negative control group has shown more bone regeneration relative to the other groups at 8 and 16 weeks. 3. All experimental groups have shown better bone regeneration relative to positive control group. 4. At 16 weeks, the first experimental group has shown more bone regeneration compared to the second experimental group. Exophytic bone formation is not good at the first and the second experimental groups compared with negative control group. But, the use of 4% inorganic polyphosphate was more effective to bone formation than the use of 8% inorganic polyphosphate. CONCLUSION: With above results, it is suggested the use of inorganic polyphosphate with vehicle under TR-ePTFE membrane.

The effect of micro-macroporous biphasic calcium phosphate incorporated with polyphosphate on exophytic bone regeneration / 대한치주과학회지

PURPOSE: In this study, the effect of micro-macroporous biphasic calcium phosphate(MBCP) incorporated with inorganic polyphosphate for bone regeneration in the calvaria of rabbit was evaluated. MATERIALS AND METHODS: The procedure of guided bone regeneration was performed with titanium reinforced expanded polytetrafluoroethylene(TR-ePTFE) membrane. Four animal groups were compared : 1) TR-ePTFE membrane for negative control group, 2) TR-ePTFE membrane filled with MBCP for positive control group, 3) TR-ePTFE membrane filled with MBCP soaked in 4% inorganic polyphosphate for experimental group I, and 4) TR-ePTFE membrane filled with MBCP soaked in 8% inorganic polyphosphate for experimental group II. RESULTS: 1. Negative control group showed the highest new bone formation at 16 weeks. 2. Positive control group showed the smallest new bone formation compared to other groups. 3. 8% inorganic polyphosphate induced more volume of bone formation, otherwise experimental group II did not show significant difference compared to negative control group. CONCLUSION: These results suggest that inorganic polyphosphate has a promoting effect on bone regeneration, possibly by enhancing osteoconductivity of the carrier and by increasing osteoinductivity of the defected alveolar bone tissue.

Histomorphometric study on effect of the polyphosphate for bone regeneration / 대한치주과학회지

In this study, author examined the effect of the concentration of the inorganic polyphosphate on the process of the bone regeneration by using the 6 weeks old rabbit with the weight of 2.0kg in average. we performed the experiment by using TR-ePTFE membrane filled with collagen immersed with 1%, 2%, and 4% of inorganic polyphosphate, respectively, after removing the proper sized cortical bones from the calvaria of rabbit. The experimental results were compared with the one of the following four groups: The control group for membrane only, experimental group I for membrane filled with collagen immersed with 1% of inorganic polyphosphate, experimental group II for membrane filled with collagen immerse with 2% of inorganic polyphosphate, experimental group III for membrane filled with collagen immersed with 4% of inorganic polyphosphate. The fragments of the tissue with membrane were obtained from each group of the sacrificed rabbits for 4 or 8 weeks sustained after surgery, were then prestained and coated. New bone formation was assessed by histomorphometric and statistical analysis. We may draw the conclusions from these experiments as following: 1. Collagen was an excellent carrier with a minimal inflammatory reaction and sustaining the form. 2. The sample of the 8th week group has shown the best bone regeneration compared with the cases of all groups including the control group. 3. The samples of collagen immersed with 2% and 4% of inorganic polyphosphate have shown more bone regeneration relative to the sample of the 1% inorganic polyphosphate. 4. The new bone regeneration was shown actively in the group for membrane filled with collagen immersed with 4% of inorganic polyphosphate. With above results, it is strongly suggested the use of inorganic polyphosphate with vehicle under TR-ePTFE membrane.

Effect of deproteinized bovine bone mineral soaked in inorganic polyphosphate on bone regeneration / 대한치주과학회지

This study was performed to evaluate the effect of deproteinized bovine bone mineral soaked in inorganic polyphosphate on bone regeneration in the calvaria of rabbit in the procedure of guided bone regeneration with titanium reinforced expanded polytetrafluoroethylene(TR-ePTFE) membrane. The rabbits were divided into four groups. Control group used TR-ePTFE membrane filled with deproteinized bovine bone mineral, experimental group I used TR-ePTFE membrane and deproteinized bovine bone mineral soaked in 4% inorganic polyphosphate, experimental group II and III used TR-ePTFE membrane and deproteinized bovine bone mineral soaked in 8% or 16% inorganic polyphosphate respectively. After decortication in the calvaria, GBR procedure was performed on 8 rabbits with only TR-ePTFE membrane or titanium reinforced ePTFE membrane filled with deproteinized bovine bone mineral soaked in inorganic polyphosphate. The animals were sacrificed at 4 weeks, and 8 weeks after the surgery. Non-decalcified specimens were processed for histologic analysis, and new bone formation was assessed by histomorphometric as well as statical analysis. 1. Both control group and experimental group demonstrated increasing of new bone formation until 8weeks. 2. At 8 weeks, experimental group I and group II showed the significant difference compared to control group in new bone formation. Especially experimental group II showed the most increasing of new bone formation. 3. The higher concentration of inorganic polyphosphate filled, the more volume of bone formation promoted, but experimental group III did not reveal significant difference compared to contol group. 4. Deproteinized bovine bone mineral did not resorbed at all until 8 weeks. These results suggest that inorganic polyphosphate has a promoting effect on bone regeneration, possibly by enhancing osteoconductivity of the carrier and by increasing osteoinductivity of the defected alveolar bone tissue, but not as we respect.