Real-time PCR assay is superior to other methods for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran

Mycoplasmas are the most important contaminants of cell cultures throughout the world. They are considered as a major problem in biological studies and biopharmaceutical economic issues. In this study, our aim was to find the best standard technique as a rapid method with high sensitivity, specificity and accuracy for the detection of mycoplasma contamination in the cell lines of the National Cell Bank of Iran. Thirty cell lines suspected to mycoplasma contamination were evaluated by five different techniques including microbial culture, indirect DNA DAPI staining, enzymatic mycoalert^® assay, conventional PCR and real-time PCR. Five mycoplasma-contaminated cell lines were assigned as positive controls and five mycoplasma-free cell lines as negative controls. The enzymatic method was performed using the mycoalert^® mycoplasma detection kit. Real-time PCR technique was conducted by PromoKine diagnostic kits. In the conventional PCR method, mycoplasma genus-specific primers were designed to analyze the sequences based on a fixed and common region on 16S ribosomal RNA with PCR product size of 425 bp. Mycoplasma contamination was observed in 60, 56.66, 53.33, 46.66 and 33.33 % of 30 different cell cultures by real-time PCR, PCR, enzymatic mycoalert^®, indirect DNA DAPI staining and microbial culture methods, respectively. The analysis of the results of the different methods showed that the real-time PCR assay was superior the other methods with the sensitivity, specificity, accuracy, predictive value of positive and negative results of 100 %. These values were 94.44, 100, 96.77, 100 and 92.85 % for the conventional PCR method, respectively. Therefore, this study showed that real-time PCR and PCR assays based on the common sequences in the 16S ribosomal RNA are reliable methods with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products.     

Biofilm control with natural and genetically-modified phages

Bacteriophages, as the most dominant and diverse entities in the universe, have the potential to be one of the most promising therapeutic agents. The emergence of multidrug-resistant bacteria and the antibiotic crisis in the last few decades have resulted in a renewed interest in phage therapy. Furthermore, bacteriophages, with the capacity to rapidly infect and overcome bacterial resistance, have demonstrated a sustainable approach against bacterial pathogens-particularly in biofilm. Biofilm, as complex microbial communities located at interphases embedded in a matrix of bacterial extracellular polysaccharide substances (EPS), is involved in health issues such as infections associated with the use of biomaterials and chronic infections by multidrug resistant bacteria, as well as industrial issues such as biofilm formation on stainless steel surfaces in food industry and membrane biofouling in water and wastewater treatment processes. In this paper, the most recent studies on the potential of phage therapy using natural and genetically-modified lytic phages and their associated enzymes in fighting biofilm development in various fields including engineering, industry, and medical applications are reviewed. Phage-mediated prevention approaches as an indirect phage therapy strategy are also explored in this review. In addition, the limitations of these approaches and suggestions to overcome these constraints are discussed to enhance the efficiency of phage therapy process. Finally, future perspectives and directions for further research towards a better understanding of phage therapy to control biofilm are recommended.     

Economics of Oil Production from Pongamia (<Emphasis Type="Italic">Millettia pinnata</Emphasis>) for Biofuel in Australia

Pongamia (Millettia pinnata) has been widely studied as a potential feedstock for biodiesel fuel, though little is known about its feasibility at a commercial level. Capital budgeting and cash flow analysis was conducted for a potential Pongamia plantation and crushing plant in Queensland, Australia. For annual seed yields ranging from 20 to 80 kg (in shell) per tree, the delivered cost of Pongamia oil was estimated to be between AUD $2.22 and AUD $0.64 per litre. The seed yield range of 20 to 80 kg per tree is roughly equivalent to between 7 and 29 t per hectare at a planting density of 357 trees per hectare. Major components of the delivered cost of (Pongamia) oil are the capital expenses of land acquisition, plantation establishment and the crushing plant construction. The major operational costs include mechanical harvesting; fertiliser; control of weed, pests and diseases; seed crushing; and freight of oil to a refinery. The cost items with the greatest volume sensitivity are the capital expenses, overheads (consisting mostly of salaries and wages of employees) and the expenses associated with harvesting and crushing operations. These costs could be significantly reduced if the seed yield could be increased. Several scenarios were tested to demonstrate the effect of seed yield and oil price on the profitability and cash flow of the Pongamia enterprise. At most plausible oil prices and seed yields, Pongamia oil is not expected to be economically viable.     

Structural insights into Rab21 GTPase activation mechanism by molecular dynamics simulations

Rab proteins belong to the family of monomeric GTPases which are involved in the cellular membrane trafficking. Rab21 protein exists in interchangeable GTP- and GDP-bound states. Rabs switch between two active and inactive conformations like other GTPases. The inactive form of Rab is bound to GDP while its active form is bounded with the GTP. Interexchange between active and inactive form is mediated by the GDP/GTP exchange factor (GEF) which catalyses the conversion from GDP-bound to GTP-bound form, thereby activating the Rab. While the GTP hydrolysis of Rabs is regulated by a GTPase-activating protein (GAP) which causes Rab inactivation. Here, we report the structural flexibility of the Rab21-GTP and Rab21-GDP complexes by docking and molecular dynamics (MD) simulations. Structural analysis of exchange mechanisms of the co-factors complexed with Rab21 reveals that Cys29, Thr33, His48, Gln78 and Lys133 are essentially important in the activation of proteins. Furthermore, a significant change in the orientation of the interacting co-factors, with slight variation in the free energy of binding was observed. Complexation of GEF with Rab21-GTP and Rab21-GDP reveal a flipping of the switchable residues. Finally, 50 ns MD simulations confirm that the GTP-bound Rab21 complex is thermodynamically more favoured than the corresponding GDP-bound complex. This study provides a detailed understanding of the structural elements involved in the conformational changes of Rab21.