Influence of hydrophilic amino acids and GC-content on expression of recombinant proteins used in vaccines against foot-and-mouth disease virus in Escherichia coli.

Epitope-based protein expression in Escherichia coli can be improved by adjusting its amino acid composition and encoding genes. To that end, we analyzed 24 recombinant epitope proteins (rEPs) that carry multiple epitopes derived from VP1 protein of foot-and-mouth disease virus. High level expression of the rEPs was attributed to a high content of Arg, Asn, Asp and Thr, a low content of Gln, Pro and Lys, a high content of hydrophilic amino acids and a higher isoelectric point value resulting from abundant Arg. It is also attributed to the appropriate guanine and cytosine content in the encoding genes. The data provide a reference for adjusting the amino acid composition in designing epitope-based proteins used in vaccines and for adjusting the synonymous codons to improve their expressions in E. coli.

Polyvinyl alcohol and polyethylene glycol form polymer bodies in the periplasm of Sphingomonads that are able to assimilate them.

Scanning electron microscopy (SEM) shows remarkable morphological surface changes in Sphingopyxis sp. 113P3 cells grown in polyvinyl alcohol (PVA) but not in Luria-Bertani medium (LB) (Hu et al. in Arch Microbiol 188: 235-241, 2007). However, transmission electron microscopy showed no surface changes in PVA-grown cells and revealed the presence of polymer bodies in the periplasm of PVA-grown cells, which were not observed in LB-grown cells. The presence of polymer bodies was supported by low-vacuum SEM observation of PVA- and LB-grown cells of strain 113P3, and the presence of similar polymer bodies was also found when Sphingopyxis macrogoltabida 103 and S. terrae were grown in polyethylene glycol (PEG). The extraction of PVA and PEG from the periplasmic fraction of cells using a modified Anraku and Heppel method and their analysis by MALDI-TOF mass spectrometry strongly suggested that the polymer bodies are composed of PVA and PEG, respectively, in Sphingopyxis sp. 113P3 (PVA degrader) and Sphingopyxis macrogoltabida 103 or S. terrae (PEG degraders). PEG-grown S. macrogoltabida 103 and S. terrae showed higher transport of (14)C-PEG 4000 than LB-grown cells. Recombinant PegB (TonB-dependent receptor-like protein consisting of a barrel structure) interacted with PEG 200, 4000 and 20000, suggesting that the barrel protein in the outer membrane contributes to the transport of PEG into the periplasm.

Chlorogenic Acid Promotes Osteogenic Differentiation of Human Dental Pulp Stem Cells Through Wnt Signaling.

Periodontal disease (PD) is one of the main causes of periodontal bone resorption and tooth loss in adults. How to repair the alveolar bone effectively has always been a challenge. This study was designed to clarify the effects and the underlying molecular mechanisms of chlorogenic acid (CGA) on osteogenic differentiation of human dental pulp stem cells (hDPSCs). In this study, we used CGA to treat hDPSCs. The osteogenic experiment showed that CGA can promote hDPSCs osteogenic differentiation. RNA-Seq and quantitative real-time polymerase chain reaction showed that CGA treatment enhanced the expression of the osteogenesis genes for frizzled-related protein (FRZB) and pyruvate dehydrogenase kinase 4 (PDK4) and inhibit the expression of the osteoclastogenesis genes such as those for asporin (ASPN) and cytokine-like 1 (CYTL1). Western blot analysis showed that besides FRZB, CGA treatment also caused reduction of both active and total ß-catenin, while increased the total calcium/calmodulin-dependent kinase II (CamKII), the phosphorylated CamKII (pCamKII) and the phosphorylated cAMP-response element-binding protein (pCREB). Likely, the increased osteogenesis was associated with reduced canonical Wnt/ß-catenin signaling but increased noncanonical Wnt/Ca2+ signaling. The results suggested that CGA can promote the osteogenic differentiation of hDPSCs by regulating Wnt signaling. These findings will serve as a foundation for further studies on how to repair defective alveolar bone for the patients with PD.

Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119.

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50 degrees C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (-G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20 degrees C to 75 degrees C (with an optimal range of 45 to 55 degrees C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C2 to C8) is p-nitrophenyl hexanoate (C6), indicating that the enzyme is an esterase rather than a lipase.

Biochemistry of microbial polyvinyl alcohol degradation.

Effect of minor chemical structures such as 1,2-diol content, ethylene content, tacticity, a degree of polymerization, and a degree of saponification of the main chain on biodegradability of polyvinyl alcohol (PVA) is summarized. Most PVA-degraders are Gram-negative bacteria belonging to the Pseudomonads and Sphingomonads, but Gram-positive bacteria also have PVA-degrading abilities. Several examples show symbiotic degradation of PVA by different mechanisms. Penicillium sp. is the only reported eukaryotic degrader. A vinyl alcohol oligomer-utilizing fungus, Geotrichum fermentans WF9101, has also been reported. Lignolytic fungi have displayed non-specific degradation of PVA. Extensive published studies have established a two-step process for the biodegradation of PVA. Some bacteria excrete extracellular PVA oxidase to yield oxidized PVA, which is partly under spontaneous depolymerization and is further metabolized by the second step enzyme (hydrolase). On the other hand, PVA (whole and depolymerized to some extent) must be taken up into the periplasmic space of some Gram-negative bacteria, where PVA is oxidized by PVA dehydrogenase, coupled to a respiratory chain. The complete pva operon was identified in Sphingopyxis sp. 113P3. Anaerobic biodegradability of PVA has also been suggested.

Proposed oxidative metabolic pathway for polypropylene glycol in Sphingobium sp. strain PW-1.

Polypropylene glycol (PPG)-assimilating Sphingobium sp. strain PW-1 was grown on 0.5% PPG 700. PPG and its metabolites were analyzed by MALDI-TOF mass spectrometry. An oxidized form of a terminal alcohol group appeared with each molecular species as a metabolite. Cell-free extract showed PPG dehydrogenase activity. In this way, the oxidative metabolic pathway for PPG was confirmed.

Cloning and expression of soluble cytochrome c and its role in polyvinyl alcohol degradation by polyvinyl alcohol-utilizing Sphingopyxis sp. strain 113P3.

The gene encoding cytocrome c in the pva operon of Sphingopyxis sp. strain 113P3 was cloned, on the basis of the sequence of the gene for cytochrome c (GenBank accession no. AB190288). The deduced amino acid sequence of the gene showed homologies (37% and 47% identities) with two cytochromes c of different origins. The recombinant cytochrome c tagged with hexahistidines was expressed in the periplasm of Escherichia coli BL21(DE3) harboring pT-GroE, which was in accordance with the localization of cytochrome c in strain 113P3; the protein was purified to homogeneity. The purified recombinant cytochrome c was a monomeric protein with a molecular weight of 16.5 kDa. The oxidized and reduced forms of the protein showed absorption maxima at 409 nm and at 414, 520 and 550 nm, respectively. The recombinant cytochrome c was fully reduced by polyvinyl alcohol (PVA), coupled with a catalytic amount (1/10 molar concentration) of the recombinant PVA dehydrogenase (PVADH) of the same origin, suggesting that the cytochrome c involved in the pva operon is a physiological primary electron acceptor for PVADH and that PVA dehydrogenation is linked with the respiratory chain in Sphingopyxis sp. strain 113P3.

[The influence of lower vertical dimension construction of complete denture on cerebral blood flow].

PURPOSE: To investigate the influence of lower vertical dimension construction on cerebral blood flow among patients with complete denture. METHODS: Ten edentulous patients were chosen and lower vertical dimension was constructed with complete denture. Transcranial Doppler ultrasonography was used to detect the average peak flow velocity, peak systolic velocity and end-diastolic peak flow velocity of the middle cerebral artery. The detection was performed before chewing, 10 minutes after chewing and 20 minutes after chewing, respectively. Before-after self control study was designed, and SPSS18.0 software package was used to analyze the data with independent samples t test and multiple comparisons and analysis of variance. RESULTS: There was no significant increase in cerebral blood flow before chewing, 10 minutes after chewing and 20 minutes after chewing in the experimental group, while blood flow velocity of the control group was significantly increased. The blood flow velocity of the experimental group 10 minutes after chewing was significantly lower than that of the control group, while no significant difference was found after chewing between the experimental group and control group 20 minutes. CONCLUSIONS: No significant increase of cerebral blood flow is detected in patients whose vertical dimension are lower when restored with complete denture during mastication.

[In vitro study on shear bond strength of veneering ceramics to zirconia].

OBJECTIVE: To investigate the shear bond strength between veneering ceramic and zirconia core in different all-ceramic systems. METHODS: Twenty disk-shaped specimens with 8 mm in diameter and 3 mm in height for each zirconia system (Lava, Cercon, IPS e.max ZirCAD, Procera) were fabricated respectively and divided into four groups: Lava group, Cercon group, IPS e.max ZirCAD group, Procera group. For each group, 10 specimens were sintered with 1 mm corresponding veneering ceramic, while the other were sintered with 2 mm corresponding veneering ceramic respectively. The shear bond strength and fracture mode of specimens were observed and determined. RESULTS: The values of shear bond strength for Lava, Cercon, IPS e.max ZirCAD and Procera were (13.82 +/- 3.71), (13.24 +/- 2.09), (6.37 +/- 4.15), (5.19 +/- 5.31) MPa in the group of 1 mm thicked veneering ceramics, respectively, while the values in the group of 2mm thicked veneering ceramics were (38.77 +/- 1.69), (21.67 +/- 3.34), (12.70 +/- 4.24), (9.94 +/- 6.67) MPa. The values of Lava and Cercon groups were significantly higher than that of IPS e.max ZirCAD and Procera groups (P < 0.05). And the values of 2 mm thicked veneering ceramic group were significantly higher than that in 1 mm thicked groups (P < 0.05). Adhesive fracture between core and veneering ceramics were observed in the fracture modes of most specimens. CONCLUSION: The shear bond strength of veneering ceramic to the zirconia framework are different from the zirconia system we chose, and the thickness of veneering ceramic has a great impact on its shear bond strength.

A recombinant truncated FMDV 3AB protein used to better distinguish between infected and vaccinated cattle.

To distinguish the antibodies induced by Foot-and-mouth disease virus (FMDV) infection from those induced by vaccination, a recombinant N-terminal truncated FMDV non-structural protein (NSP) of 3AB, designated as r3aB, was constructed by deleting 80 amino acids displayed about 30% homology to transposase IS4 family protein of Escherichia coli, expressed in E. coli BL21 (DE3) and then purified. The r3aB was majorly expressed in soluble fraction and presented as homogeneous monomers after purification. Using r3aB as coating antigen, an indirect ELISA was established to specifically identify antibodies induced by FMDV infection but not those induced by vaccination. Compared with 3AB, r3aB was more specific to catch antibodies against NSP. The performance of this assay was validated by two commercial FMDV NSP ELISA kits. The result suggested that the r3aB coated ELISA could be developed into a kit to better distinguish between infected and vaccinated cattle.

[An in vitro study of retentive force and deformation of resin clasp].

OBJECTIVE: To study the retentive force and deformation of acetal resin clasp. METHODS: 40 premolars and 40 molars were cast respectively. Undercut of 0.25 mm or 0.50 mm depth were measured for each with undercut gage. According to the type of abutment and the depth of undercut, the specimens were divided into 4 groups: Premolars with 0.25 mm undercut, premolars with 0.50 mm undercut, molars with 0.25 mm undercut and molars with 0.50 mm undercut, 20 specimens each group. 10 three-arm clasps with resin and Co-Cr alloy were fabricated in each group, respectively. The clasps were set into the corresponding abutments and soaked in distilled water. The retentive force of the clasps when 0, 720, 1440, 2160, 2880, 3600, 4320 consecutive times of setting in and removing out from the abutments were measured. The distance between the tips of retentive arm and resistant arm after 0 and 4320 cycles were recorded. RESULTS: 1) The mean retentive force of resin clasps (1.69 N) was significantly lower than that of Co-Cr clasps (5.87 N) (P<0.01). With the same factors, the retentive force of resin clasps were significantly less than that of Co-Cr clasps (P<0.01). The retentive force of molar clasps were significantly lower than that of premolar models (P<0.01). The retentive force of 0.25 mm undercut clasps were significantly lower than that of 0.50 mm undercut clasps (P<0.01). With increasing time of the cycles, the retentive force of Co-Cr clasps significantly reduced (P<0.01), but the retentive force of resin clasps didn't change significantly (P>0.05). 2) After 4320 times, the distance between the tips of retentive arm and resistant arm of Co-Cr clasps increased significantly (P<0.05), but the distance between the tips of resin clasps didn't change significantly (P>0.05). CONCLUSION: The retentive force and deformation of the resin clasp are significantly lower than those of Co-Cr clasp.

The pva operon is located on the megaplasmid of Sphingopyxis sp. strain 113P3 and is constitutively expressed, although expression is enhanced by PVA.

The upstream and downstream regions of the tentative pva operon including genes encoding oxidized polyvinyl alcohol (PVA) hydrolase (oph), PVA dehydrogenase (pvaA) and cytochrome c (cytC) from Sphingopyxis sp. strain 113P3 were sequenced. The resultant 7.9 kb sequence contained orf1 in the upstream region and orf2 and orf3 in the downstream region. Reverse transcription-polymerase chain reaction (PCR) analyses revealed that the intergenic regions between orf1 and oph or between cytC and orf2 were expressed neither in PVA medium nor glucose medium, indicating that the pva operon consists of three genes. A transcription start site was determined by 5'-rapid amplification of cDNA ends to be 428 bp upstream of the start codon of the oph. The stop codon of cytC was followed by a sequence of inverted repeats that could function as a factor-independent transcription terminator. Strain 113P3 had one megaplasmid including the pva operon. Northern blot hybridization for the three genes revealed that mRNA size was approximately 3 to 4 kb and expression was elevated in PVA medium compared to glucose medium.

Isolation of bacteria able to grow on both polyethylene glycol (PEG) and polypropylene glycol (PPG) and their PEG/PPG dehydrogenases.

Two bacterial consortia growing on a random copolymer of ethylene glycol and propylene glycol units were obtained by enrichment cultures from various microbial samples. Six major strains included in both consortia were purified and identified as Sphingomonads, Pseudomonas sp. and Stenotrophomonas maltophilia. Three of them (Sphingobium sp. strain EK-1, Sphingopyxis macrogoltabida strain EY-1, and Pseudomonas sp. strain PE-2) utilized both PEG and polypropylene glycol (PPG) as a sole carbon source. Four PEG-utilizing bacteria had PEG dehydrogenase (PEG-DH) activity, which was induced by PEG. PCR products from DNA of these bacteria generated with primers designed from a PEG-DH gene (AB196775 for S. macrogoltabida strain 103) indicated the presence of a sequence that is the homologous to the PEG-DH gene (99% identity). On the other hand, five PPG-utilizing bacteria had PPG dehydrogenase (PPG-DH) activity, but the activity was constitutive. PCR of a PPG-DH gene was performed using primers designed from a polyvinyl alcohol dehydrogenase (PVA-DH) gene (AB190288 for Sphingomonas sp. strain 113P3) because a PPG-DH gene has not been cloned yet, but both PPG-DH and PVA-DH were active toward PPG and PVA (Mamoto et al. 2006). PCR products of the five strains did not have similarity to each other or to oxidoreductases including PVA-DH.

Cell surface structure enhancing uptake of polyvinyl alcohol (PVA) is induced by PVA in the PVA-utilizing Sphingopyxis sp. strain 113P3.

Polyvinyl alcohol (PVA)-utilizing Sphingopyxis sp. 113P3 (re-identified from Sphingomonas sp. 113P3) removed almost 0.5% PVA from culture supernatants in 4 days. Faster degradation of 0.5% PVA was performed by the periplasmic fraction. The average molecular size of PVA in the culture supernatant or cell-bound PVA was gradually shifted higher, suggesting that lower molecular size molecules are degraded faster. Depolymerized products were found in neither the culture supernatant nor the cell-bound fraction; however they were recovered from the periplasmic fraction. As extracellular or cell-associated PVA oxidase activity was almost undetectable in strain 113P3, degradation of PVA must be performed by periplasmic PVA dehydrogenase after uptake into the periplasm. Following the consumption of PVA, a dent appeared on the cell surface on day 2 and increased in size and depth for 4 days and was maintained for 8 days. Ultrastructural change on the cell surface was only observed in PVA medium, but not in nutrient broth (NB), suggesting that the change is induced by PVA. Fluorescein-4-isothiocyanate-labeled PVA was bound more to cells grown in PVA than to cells grown in NB. No binding was found with PVA-grown cells treated with formaldehyde. Thus, a dent on the cell surface seems to be related to the uptake of PVA.

Coating thickness of magnetic iron oxide nanoparticles affects R2 relaxivity.

PURPOSE: To evaluate the effect of coating thickness on the relaxivity of iron oxide nanoparticles. MATERIALS AND METHODS: Monocrystalline superparamagnetic iron oxide nanoparticles (MIONs), coated with a polyethylene glycol (PEG)-modified, phospholipid micelle coating, with different PEG molecular weights, were prepared. The particle diameters were measured with dynamic light scattering (DLS) and electron microscopy (EM). The R1 and R2 of MIONs were measured using a bench-top nuclear magnetic resonance (NMR) relaxometer. pH was varied for some measurements. Monte Carlo simulations of proton movement in a field with nanometer-sized magnetic inhomogeneities were performed. RESULTS: Increasing the molecular weight of the PEG portion of the micelle coating increased overall particle diameter. As coating thickness increases, the R2 decreases and the R1 increases. Changing pH has no effect on relaxivity. The Monte Carlo simulations suggest that the effect of coating size on R2 relaxivity is determined by two competing factors: the physical exclusion of protons from the magnetic field and the residence time for protons within the coating zone. CONCLUSION: Coating thickness can significantly impact the R2, and the R2/R1 ratio, of a MION contrast agent. An understanding of the relationship between coating properties and changes in relaxivity is critical for designing magnetic nanoparticle probes for molecular imaging applications using MRI.

Long-circulating liposomal contrast agents for magnetic resonance imaging.

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a dynamic technique for imaging vasculature. However, the currently used gadolinium (Gd) chelates, such as Gd-DTPA, restrict the time window for image acquisition due to their rapid elimination from blood and their rapid diffusion into the extravascular space, which prevents their use in steady-state imaging, particularly for MR angiography (MRA). The goal of this study was to prepare long-circulating polyethylene glycol-bearing ((PEG)ylated) liposomes encapsulating Gd chelate, and characterize and demonstrate their utility for MRA. The liposomes were prepared by hydrating a mixture of lipids with gadodiamide (Omniscan). The liposomes were sized down to around 100 nm by extruder and exhaustively dialysed to remove the unencapsulated gadodiamide. The Gd liposomes exhibited a significant sustained (>4 hr) contrast enhancement of the vasculature with improved spatial details in a rat model with little leakage relative to Gd-DTPA controls as shown by MRI. We suggest that such long-circulating liposomal formulations allow for high spatial resolution imaging without the confounding effects of clearance and extravascular diffusion of the agent complicating the data and image analysis.