Evaluating the efficiency of phiC31 integrase‐mediated monoclonal antibody expression in CHO cells

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site‐specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO‐S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570–1576, 2016     

Genetic Variation and Association Analysis of the SSR Markers Linked to the Major Drought-Yield QTLs of Rice

Drought is one of the major abiotic stresses, which hampers the production of rice worldwide. Informative molecular markers are valuable tools for improving the drought tolerance in various varieties of rice. The present study was conducted to evaluate the informative simple sequence repeat (SSR) markers in a diverse set of rice genotypes. The genetic diversity analyses of the 83 studied rice genotypes were performed using 34 SSR markers closely linked to the major quantitative trait loci (QTLs) of grain yield under drought stress (qDTYs). In general, our results indicated high levels of polymorphism. In addition, we screened these rice genotypes at the reproductive stage under both drought stress and nonstressful conditions. The results of the regression analysis demonstrated a significant relationship between 11 SSR marker alleles and the plant paddy weight under stressful conditions. Under the nonstressful conditions, 16 SSR marker alleles showed a significant correlation with the plant paddy weight. Finally, four markers (RM279, RM231, RM166, and RM231) demonstrated a significant association with the plant paddy weight under both stressful and nonstressful conditions. These informative-associated alleles may be useful for improving the crop yield under both drought stress and nonstressful conditions in breeding programs.     

The effect of alumina nanoparticles on the thermal properties of PMMA: a molecular dynamics simulation

Metal oxides, as one of the most promising flame retardant additives, improve the fire retardant and the thermal stability properties of polymers. In the present study, molecular dynamics (MD) simulations based on the united atom model were applied to study the effect of alumina nanoparticles on the density, thermal conductivity, heat capacity, and thermal diffusivity of isotactic poly(methyl methacrylate) (is-PMMA). Thermal diffusivity of PMMA and PMMA/alumina nanocomposite were investigated through calculating thermal conductivity, density and heat capacity in the range of 300–700?K. Heat capacity can be calculated using fluctuations properties of energy. Thermal conductivity was calculated through the nonequilibrium molecular dynamics (NEMD) simulation by Fourier’s law approach. Our results show that the addition of alumina nanoparticles decreases the heat capacity and increases the glass transition temperature (T_g), thermal conductivity and thermal diffusivity of the PMMA. Therefore, the addition of alumina nanoparticles to PMMA improves the fire retardancy of the polymer. In addition, we illustrate the links between the intermolecular and bulk properties of PMMA in the presence of the alumina nanoparticles.     

Simulation-Based Exploration of Quorum Sensing Triggered Resistance of Biofilms to Antibiotics

We present a mathematical model of biofilm response to antibiotics, controlled by a quorum sensing system that confers increased resistance. The model is a highly nonlinear system of partial differential equations that we investigate in computer simulations. Our results suggest that an adaptive, quorum sensing-controlled, mechanism to switch between modes of fast growth with little protection and protective modes of slow growth may confer benefits to biofilm populations. It enhances the formation of micro-niches in the inner regions of the biofilm in which bacteria are not easily reached by antibiotics. Whereas quorum sensing inhibitors can delay the onset of increased resistance, their advantage is lost after up-regulation. This emphasizes the importance of timing for treatment of biofilms with antibiotics.     

Efficient Parthenogenesis Induction and In Vitro Haploid Plant Regeneration in Cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) Using Putrescine,Spermidine, and Cycocel

In this study, the effect of spraying mother plants with various levels of putrescine, spermidine, and cycocel (each at 0, 50, 500, and 5000 mg/l) were assessed on the frequency of haploid embryos produced from unfertilized ovaries and subsequent regeneration of derived embryos. Significantly higher haploid embryos were obtained when mother plants were sprayed with putrescine at 500 mg/l (5.2 embryos/fruit), spermidine at 50 mg/l (4.8 embryos/fruit), and cycocel at 50 mg/l (5.2 embryos/fruit) as compared to the control (without spraying, 3.2 embryos/fruit). However, embryogenesis induction was decreased drastically as the concentration of all the three compounds tested was increased and the lowest haploid embryos were observed when 5000 mg/l of spermidine (0.4 embryos/fruit) or cycocel (2.0 embryos/fruit) were applied. Only spermidine at 50 mg/l led to 100% regeneration into fully developed plantlets. The seed setting and size of fruits were also affected by polyamines and cycocel applications. Ploidy analysis using a flow cytometer indicated that all regenerated plantlets contain the gametic chromosome number (n?=?x?=?7) of parental plants and the results of chromosome counting also confirmed the haploid nature of regenerated plantlets. It can be concluded that the induction of haploid embryogenesis from unfertilized ovaries after pollination with irradiated pollen and subsequent conversion of derived embryos into the plantlets could be improved in Cucumis sativus L. by applying appropriate levels of putrescine, spermidine, and cycocel.