Utilization of the recombinant human β-carotene-15,15′-monooxygenase gene in <Emphasis Type="Italic">Escherichia coli</Emphasis> and mammalian cells

In animals, β-carotene 15,15′-monooxygenase (BCMO) is the key enzyme involved in the metabolism of plant β-carotene to retinal. In the present study, we utilized β-carotene-producing Escherichia coli to screen for mutants with higher BCMO activity which was monitored by color changes derived from β-carotene cleavage. Recombinant wild-type and T381L mutant BCMO proteins were purified to near homogeneity in E. coli, and their enzymatic activities were determined by HPLC analysis. The catalytic efficiency for β-carotene and retinal production of the mutant were 1.5-fold and 1.7-fold higher than those of wild-type, respectively. Further BCMO function in mammalian cells was analyzed by a retinoic acid receptor reporter assay, which responds to the metabolic conversion of β-carotene to retinoic acid in vivo. Overall, these tools can be used to screen more active BCMO for the industrial and pharmacological purpose of retinal production from β-carotene.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

Activation of cytotoxic T lymphocytes against CML28-bearing tumors by dendritic cells transduced with a recombinant adeno-associated virus encoding the CML28 gene

Induction of anti-tumor immune responses by dendritic cells (DCs) transduced with a recombinant adeno-associated virus type 2 (rAAV2) encoding tumor antigens is considered a promising approach for cancer vaccine development. CML28, a novel antigen with the properties of cancer/testis (CT) antigens, is an attractive target for antigen-specific immunotherapy. Here we investigated the feasibility of inducing CML28-specific cytotoxic T lymphocyte (CTL) responses using DCs transduced with the rAAV2 vectors containing the CML28 gene (rAAV/CML28). Using an adenovirus-free packaging system, rAAV/CML28 was generated. The transduction efficiency of rAAV/CML28 in DCs increased in a multiplicity of infection (MOI)-dependent manner. The rAAV/CML28 transduction did not impair DC maturation, but even enhanced the CD80 expression. The rAAV/CML28-transduced DCs induced CML28-specific CTLs which exhibited a MHC class I-mediated antigen-specific lytic activity against CML28-bearing tumor cell lines (HepG2 and MCF-7) as well as the primary leukemia blasts. These findings suggest that rAAV/CML28-transduced DCs vaccine may serve as a feasible approach for the treatment of CML28-associated cancers.     

Treatment of odorous volatile fatty acids using a biotrickling filter

In this study, a novel fibrous bioreactor was developed for treating odorous compounds present in contaminated air. The first stage of this work was a preliminary study which aimed at investigating the feasibility of using the fibrous bioreactor for the removal of malodorous volatile fatty acids (VFA) that is a common odorous contaminant generated from anaerobic degradation of organic compounds. The kinetics of microbial growth and VFA degradation in the selected culture, and the performance of the submerged bioreactor at different VFA mass loadings were studied. Above 95% of VFA removal efficiencies were achieved at mass loadings up to 22.4 g/m(3)/h. In the second stage, the odour treatment process was scaled up with system design and operational considerations. A trickling biofilter with synthetic fibrous packing medium was employed. The effects of inlet VFA concentration and empty bed retention time (EBRT) on the process performance were investigated. The bioreactor was effective in removing VFA at mass loadings up to 32 g/m(3)/h, beyond which VFA started to accumulate in the recirculation liquid, indicating the biofilm was unable to degrade all of the VFA introduced. Although VFA accumulated in the liquid phase, the removal efficiency remained above 99%. This suggested that the biochemical reaction rather than gas-liquid mass transfer was the limiting step of the treatment process. In addition, the biotrickling filter was stable for long-term operation with relatively low and steady pressure drop, no clogging and degeneration of the packing material occurred during the four-month study.     

Performance study of vegetated sequencing batch coal slag bed treating domestic wastewater in suburban area

A practical and affordable wastewater treatment system serving small community in suburban areas was studied. The system was a vegetated sequencing batch coal slag bed integrated with the rhythmical movement of wastewater and air like that of a sequencing batch reactor. The removal mechanisms capitalized on the pollutant removal process in conventional constructed wetland. Cyperus alternifolius was planted into the coal slag bed to form a novel plant-soil-microbial interactive system. Nutrients in the domestic wastewater, which cause environmental nuisance like eutrophication, were targeted to be eliminated by the process design. Operated with the contact time of 18 h, the treatment systems achieved around 60% removal efficiency for carbonaceous matters. The removals of ammonia nitrogen and phosphorus were about 50% and 40%, respectively, while the removal of total suspended solids was approaching 80%. From the current study, the construction cost of the vegetated sequencing batch coal slag bed was 256 RMB/m3 and the operation cost was 0.13 RMB/m3. With the advantages of ease of operation, low costs, desirable treatment efficiency and aesthetic value, the vegetated sequencing batch coal slag bed is proposed to be an alternative for onsite domestic wastewater treatment in suburban areas.     

Lactobacillus plantarum USM8613 Aids in Wound Healing and Suppresses Staphylococcus aureus Infection at Wound Sites

Probiotics and Antimicrobial Proteins - This study aimed to elucidate the targets and mechanisms of anti-staphylococcal effects from bioactive metabolites produced by lactic acid bacteria. We aimed...     

Combined Stimulation of IL-2 and 4-1BB Receptors Augments the Antitumor Activity of E7 DNA Vaccines by Increasing Ag-Specific CTL Responses

Human papillomavirus (HPV) infection is a major cause of cervical cancer. Here, we investigate whether concurrent therapy using HPV E7 DNA vaccines (pE7) plus IL-2 vs. IL-15 cDNA and anti-4-1BB Abs might augment antitumor activity against established tumors. IL-2 cDNA was slightly better than IL-15 cDNA as a pE7 adjuvant. Co-delivery of pE7+IL-2 cDNA increased tumor cure rates from 7% to 27%, whereas co-delivery of pE7+IL-2 cDNA with anti-4-1BB Abs increased tumor cure rates from 27% to 67% and elicited long-term memory responses. This increased activity was concomitant with increased induction of Ag-specific CTL activity and IFN-γ responses, but not with Ag-specific IgG production. Moreover, the combined stimulation of IL-2 and 4-1BB receptors with rIL-2 and anti-4-1BB Abs resulted in enhanced production of IFN-γ from Ag-specific CD8+ T cells. However, this effect was abolished by treatment with anti-IL-2 Abs and 4-1BB-Fc, suggesting that the observed effect was IL-2- and anti-4-1BB Ab-specific. A similar result was also obtained for Ag-specific CTL activity. Thus, these studies demonstrate that combined stimulation through the IL-2 and 4-1BB receptors augments the Ag-specific CD8+ CTL responses induced by pE7, increasing tumor cure rates and long-term antitumor immune memory. These findings may have implications for the design of DNA-based therapeutic vaccines against cancer.     

Esterase polymorphism and sensitivity to dursban organophosphorus insecticide in Culex pipiens pipiens populations

Esterase polymorphism and Dursban (O,O-dimethyl-2-pyridylphosphorothioate) sensitivity have been investigated in 12 natural populations and three laboratory strains of Culex pipiens pipiens. This mosquito has two esterase loci, Est-1 and Est-2, which were shown to code esterases of the B group (aliesterases) but not cholinesterases. No correlation between Est-1 polymorphism and Dursban sensitivity was found, but the increase of the Est-2(0.64) allele in the populations less sensitive to Dursban was highly significant (r = -0.9850 for 6 df).