Genome-wide RNAi screen for improved functional expression of recombinant proteins from HEK 293 cells

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. 56 genes whose silencing caused the greatest improvement in the luciferase expression were found to be part of several different pathways that are associated with spliceosome formation/mRNA processing, transcription, metabolic process, transport and protein folding. 10 genes whose downregulation significantly enhanced the protein expression were validated by their silencing effect on four different recombinant proteins. Among the validated genes, the gene encoding the ornithine decarboxylase antizyme1- was selected for detailed investigation, since its silencing improved the reporter protein production without affecting cell viability. Silencing this gene caused the increase of the ornithine decarboxylase enzyme and the cellular levels of putrescine and spermidine, and indicated that increased cellular polyamines enhanced luciferase expression without affecting its transcription. The study shows that this gene is a novel target for improving expression of recombinant proteins. The genome-scale screening demonstrated in this work can establish the foundation for targeted design of an efficient mammalian cell platform for different biotechnological applications

Degree-constrained Subgraph Reconfiguration is in P

The degree-constrained subgraph problem asks for a subgraph of a given graph such that the degree of each vertex is within some specified bounds. We study the following reconfiguration variant of this problem: Given two solutions to a degree-constrained subgraph instance, can we transform one solution into the other by adding and removing individual edges, such that each intermediate subgraph satisfies the degree constraints and contains at least a certain minimum number of edges? This problem is a generalization of the matching reconfiguration problem, which is known to be in P. We show that even in the more general setting the reconfiguration problem is in P.Comment: Full version of the paper published at Mathematical Foundations of Computer Science (MFCS) 201

Maintenance of Strongly Connected Component in Shared-memory Graph

In this paper, we present an on-line fully dynamic algorithm for maintaining strongly connected component of a directed graph in a shared memory architecture. The edges and vertices are added or deleted concurrently by fixed number of threads. To the best of our knowledge, this is the first work to propose using linearizable concurrent directed graph and is build using both ordered and unordered list-based set. We provide an empirical comparison against sequential and coarse-grained. The results show our algorithm's throughput is increased between 3 to 6x depending on different workload distributions and applications. We believe that there are huge applications in the on-line graph. Finally, we show how the algorithm can be extended to community detection in on-line graph.Comment: 29 pages, 4 figures, Accepted in the Conference NETYS-201

Mixed adsorption and surface tension prediction of nonideal ternary surfactant systems

To deal with the mixed adsorption of nonideal ternary surfactant systems, the regular solution approximation for nonideal binary surfactant systems is extended and a pseudo-binary system treatment is also proposed. With both treatments, the compositions of the mixed monolayer and the solution concentrations required to produce given surface tensions can be predicted based only on the gamma-LogC curves of individual surfactants and the pair interaction parameters. Conversely, the surface tensions of solutions with different bulk compositions can be predicted by the surface tension equations for mixed surfactant systems. Two ternary systems: SDS/Hyamine 1622/AEO7, composed of homogeneous surfactants, and AES/DPCl/AEO9, composed of commercial surfactants, in the presence of excess NaCl, are examined for the applicability of the two treatments. The results show that, in general, the pseudo-binary system treatment gives better prediction than the extended regular solution approximation, and the applicability of the latter to typical anionic/cationic/nonionic nonideal ternary surfactant systems seems to depend on the combined interaction parameter, $${\mathop {(\beta }\nolimits_{an}^s } + {\mathop \beta \nolimits_{cn}^s })/2 - {\mathop \beta \nolimits_{ac}^s }/4$$ : the more it deviates from zero, the larger the prediction difference. If $${\mathop {(\beta }\nolimits_{an}^s } + {\mathop \beta \nolimits_{cn}^s })/2 - {\mathop \beta \nolimits_{ac}^s }/4$$ rarr0, good agreements between predicted and experimental results can be obtained and both treatments, though differently derived, are interrelated and tend to be equivalent

Glucose uptake regulation in E. coli by the small RNA SgrS: comparative analysis of E. coli K-12 (JM109 and MG1655) and E. coli B (BL21)

<p>Abstract</p> <p>Background</p> <p>The effect of high glucose concentration on the transcription levels of the small RNA SgrS and the messenger RNA ptsG, (encodin<it>g </it>the glucose transporter IICB^Glc), was studied in both <it>E. coli </it>K-12 (MG1655 and JM109) and <it>E. coli </it>B (BL21). It is known that the transcription level of <it>sgrS </it>increases when <it>E. coli </it>K-12 (MG1655 and JM109) is exposed to the non-metabolized glucose alpha methyl glucoside (αMG) or when the bacteria with a defective glycolysis pathway is grown in presence of glucose. The increased level of sRNA SgrS reduces the level of the ptsG mRNA and consequently lowers the level of the glucose transporter IICB^Glc. The suggested trigger for this action is the accumulation of the corresponding phospho-sugars.</p> <p>Results</p> <p>In the course of the described work, it was found that <it>E. coli </it>B (BL21) and <it>E. coli </it>K-12 (JM109 and MG1655) responded similarly to αMG: both strains increased <it>SgrS </it>transcription and reduced <it>ptsG </it>transcription. However, the two strains reacted differently to high glucose concentration (40 g/L). <it>E. coli </it>B (BL21) reacted by increasing <it>sgrS </it>transcription and reducing <it>ptsG </it>transcription while <it>E. coli </it>K-12 (JM109 and MG1655) did not respond to the high glucose concentration, and, therefore, transcription of <it>sgrS </it>was not detected and ptsG mRNA level was not affected.</p> <p>Conclusions</p> <p>The results suggest that <it>E. coli </it>B (BL21) tolerates high glucose concentration not only by its more efficient central carbon metabolism, but also by controlling the glucose transport into the cells regulated by the sRNA SgrS, which may suggest a way to control glucose consumption and increase its efficient utilization.</p

Efficient Implementation of a Synchronous Parallel Push-Relabel Algorithm

Motivated by the observation that FIFO-based push-relabel algorithms are able to outperform highest label-based variants on modern, large maximum flow problem instances, we introduce an efficient implementation of the algorithm that uses coarse-grained parallelism to avoid the problems of existing parallel approaches. We demonstrate good relative and absolute speedups of our algorithm on a set of large graph instances taken from real-world applications. On a modern 40-core machine, our parallel implementation outperforms existing sequential implementations by up to a factor of 12 and other parallel implementations by factors of up to 3

Expression of human α1-proteinase inhibitor in Aspergillus niger

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog

<p>Abstract</p> <p>Background</p> <p>Genomics tools, particularly DNA microarrays, have found application in a number of areas including gene discovery and disease characterization. Despite the vast utility of these tools, little work has been done to explore the basis of distinct cellular properties, especially those important to biotechnology such as growth. And so, with the intent of engineering cell lines by manipulating the expression of these genes, anchorage-independent and anchorage-dependent HeLa cells, displaying markedly different growth characteristics, were analyzed using DNA microarrays.</p> <p>Results</p> <p>Two genes, cyclin-dependent kinase like 3 (<it>cdkl3</it>) and cytochrome c oxidase subunit (<it>cox15</it>), were up-regulated in the faster growing, anchorage-independent (suspension) HeLa cells relative to the slower growing, anchorage-dependent (attached) HeLa cells. Enhanced expression of either gene in the attached HeLa cells resulted in elevated cell proliferation, though insertion of <it>cdkl3 </it>had a greater impact than that of <it>cox15</it>. Moreover, flow cytometric analysis indicated that cells with an insert of <it>cdkl3 </it>were able to transition from the G0/G1 phases to the S phase faster than control cells. In turn, expression of <it>cox15 </it>was seen to increase the maximum viable cell numbers achieved relative to the control, and to a greater extent than <it>cdkl3</it>. Quantitatively similar results were obtained with two Human Embryonic Kidney-293 (HEK-293) cell lines and a Chinese Hamster Ovary (CHO) cell line. Additionally, HEK-293 cells secreting adipocyte complement-related protein of 30 kDa (acrp30) exhibited a slight increase in specific protein production and higher total protein production in response to the insertion of either <it>cdkl3 </it>or <it>cox15</it>.</p> <p>Conclusion</p> <p>These results are consistent with previous studies on the functionalities of <it>cdkl3 </it>and <it>cox15</it>. For instance, the effect of <it>cdkl3 </it>on cell growth is consistent with its homology to the <it>cdk3 </it>gene which is involved in G1 to S phase transition. Likewise, the increase in cell viability due to <it>cox15 </it>expression is consistent with its role in oxidative phosphorylation as an assembly factor for cytochrome c oxidase and its involvement removing apoptosis-inducing oxygen radicals. Collectively, the present study illustrates the potential of using microarray technology to identify genes influential to specific cellular processes with the possibility of engineering cell lines as desired to meet production needs.</p

Improving production of retroviral vector from Pg13 cells for T cell therapy

Adoptive T-Cell therapy is a growing field for cancer treatment using the patient’s immune system to battle the cancer cells. Tumor specific T cells are either isolated from a tumor or created by modifying the T cells and after expansion are administered to the patient. The modifications include adding specific T cell receptors (TCR) or chimeric antigen receptors (CAR) by way of retroviral vector, lentiviral vector, or other method. One method is to use PG13 cells, which are derivatives of NIH3T3 mouse fibroblasts, to stably produce a retroviral vector that is used to transduce the T cell. PG13 cells are anchorage dependent cells that grow in roller bottles or cell factories to produce the viral vector and recently in a fixed bed bioreactor. To improve the production of the viral vector we explore the possibility of its production using PG13 cells grown on microcarriers in a bioreactor. Microcarriers are small, approximately 100-300 µm, charged beads that support the attachment of the cells and are suspended in the growth media in the bioreactor that provide controlled growth conditions. In this way parameters, such as oxygen concentration, pH, and nutrient are monitored and controlled. The result is higher cell concentration and consequently virus titer. There was no effect on the specific virus titer or the efficacy of the vector in transducing t cells indicating that using microcarriers in a bioreactor is a good method for scaling up stable production of gamma retroviral vector in PG13 cells

Effect of over expressing protective antigen on global gene transcription in Bacillus anthracis BH500

Protective antigen (PA) of Bacillus anthracis is being considered as a vaccine candidate against anthrax and its production has been explored in several heterologous host systems. Since the expression approaches tested, introduced adverse issues such as inclusion body formation and endotoxin contamination, the production from B. anthracis is presently considered as a preferred method. In this presentation we will report on the effect of protective antigen expression on the metabolism of the producing train B. anthracis, BH500, by comparing it with a control strain carrying an empty plasmid. The two strains were grown in a bioreactor and RNA-seq analysis of the producing and non-producing strain was performed. Several differences were observed, especially significant were the following: the strain expressing rPA showed increased transcription of sigL, the gene encoding RNA polymerase σ54, sigB, the general stress transcription factor gene and its regulators rsbW and rsbV, as well as the global regulatory repressor ctsR. At the same time there were also decreased expression of intracellular heat stress related genes such as groL, groES, hslO, dnaJ, and dnaK and increased expression of extracellular chaperons csaA and prsA2. Additionally, major central metabolism genes belonging to TCA, glycolysis, PPP, and amino acids biosynthesis were up-regulated in the PA-producing strain which was associated with decreased specific growth rates. The information and the observation acquired from this study will be presented together with possible approaches to create a better producing strain