Improved Compact Visibility Representation of Planar Graph via Schnyder's Realizer

Let $G$ be an $n$-node planar graph. In a visibility representation of $G$, each node of $G$ is represented by a horizontal line segment such that the line segments representing any two adjacent nodes of $G$ are vertically visible to each other. In the present paper we give the best known compact visibility representation of $G$. Given a canonical ordering of the triangulated $G$, our algorithm draws the graph incrementally in a greedy manner. We show that one of three canonical orderings obtained from Schnyder's realizer for the triangulated $G$ yields a visibility representation of $G$ no wider than $\frac{22n-40}{15}$. Our easy-to-implement O(n)-time algorithm bypasses the complicated subroutines for four-connected components and four-block trees required by the best previously known algorithm of Kant. Our result provides a negative answer to Kant's open question about whether $\frac{3n-6}{2}$ is a worst-case lower bound on the required width. Also, if $G$ has no degree-three (respectively, degree-five) internal node, then our visibility representation for $G$ is no wider than $\frac{4n-9}{3}$ (respectively, $\frac{4n-7}{3}$). Moreover, if $G$ is four-connected, then our visibility representation for $G$ is no wider than $n-1$, matching the best known result of Kant and He. As a by-product, we obtain a much simpler proof for a corollary of Wagner's Theorem on realizers, due to Bonichon, Sa\"{e}c, and Mosbah.Comment: 11 pages, 6 figures, the preliminary version of this paper is to appear in Proceedings of the 20th Annual Symposium on Theoretical Aspects of Computer Science (STACS), Berlin, Germany, 200

Orderly Spanning Trees with Applications

We introduce and study the {\em orderly spanning trees} of plane graphs. This algorithmic tool generalizes {\em canonical orderings}, which exist only for triconnected plane graphs. Although not every plane graph admits an orderly spanning tree, we provide an algorithm to compute an {\em orderly pair} for any connected planar graph $G$, consisting of a plane graph $H$ of $G$, and an orderly spanning tree of $H$. We also present several applications of orderly spanning trees: (1) a new constructive proof for Schnyder's Realizer Theorem, (2) the first area-optimal 2-visibility drawing of $G$, and (3) the best known encodings of $G$ with O(1)-time query support. All algorithms in this paper run in linear time.Comment: 25 pages, 7 figures, A preliminary version appeared in Proceedings of the 12th Annual ACM-SIAM Symposium on Discrete Algorithms (SODA 2001), Washington D.C., USA, January 7-9, 2001, pp. 506-51

Enhancing crispr-mediated CHO cell antibody productivity through concentrated fed- batch or continuous perfusion

Integrated continuous bioprocessing technology has high productivity and cost saving benefit, which combines the upstream (Cell culture) and downstream (Purification) processing. The continuous bioprocessing based biopharmaceutical manufacturing is more profitable than traditional batch/fed-batch processing in increasing quality and quantity. The goal of this study focuses on the upstream continuous process development with crispr-mediated targeted gene integration CHO cell line producing monoclonal antibody. In the upstream processing, we developed concentrated fed-batch culture (CFB) and high density perfusion culture in 2-5 L bioreactor with a cell retention device (alternating tangential flow, ATF). With our concentrated fed-batch culture system, the VCD achieved 8.4x107 cells/m in 11days operation with 1VVD producing antibody 3.3-fold that of fed-batch culture system; in the high density perfusion culture system, the VCD achieved 5x107 cells/ml in 28 days operation with 1 VVD producing antibody greater than 1g/L/day with on the Day10 and keep the cell density, viability and productivity more than 1 month

Current Status of Antimicrobial Resistance in Taiwan

While some trends in antimicrobial resistance rates are universal, others appear to be unique for specific regions. In Taiwan, the strikingly high prevalence of resistance to macrolides and streptogramin in clinical isolates of gram-positive bacteria correlates with the widespread use of these agents in the medical and farming communities, respectively. The relatively low rate of enterococci that are resistant to glycopeptide does not parallel the high use of glycopeptides and extended-spectrum beta-lactams in hospitals. The evolving problem of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae isolates is substantial, and some unique enzymes have been found. Recently, some gram-negative bacteria (e.g., Pseudomonas aeruginosa and Acinetobacter baumannii) that are resistant to all available antimicrobial agents including carbapenems have emerged

Bioprocess intelligent for the improvements and prediction on fed-batch cell culture in bioreactor

With advances in biotechnology, the antibody productivity is not the only issue in biopharmaceutical manufacturing; moreover, how to control the quality and quantity of antibody production in bioprocess has become prominent. In typical fed-batch cell culture, it is not easy to control the dynamic cultivation and feeding conditions. The study is to present an intelligent bioprocess make use of design of experiments (DOE) and multivariate data analysis (MVDA). In the culture medium optimization, we performed the medium screening with the DOE method in shake spin tubes and shake flasks. DOE provides a cost-effective methodology for medium development and optimization, and furthermore we utilized multivariate data analysis methodology to build up the fed-batch intelligence bioprocess in 5 L bioreactor. We analyzed mass data transferred from 5 L bioreactor process and established the fed-batch culture model with SIMCA software. Combination of the batch process and big data sets, we can be easy to do batch-to-batch comparison, cell culture profile prediction, and key parameter finding to improve the process performance with more steady product quality and less error. In this fed-batch culture model, we achieved more than 5 g/L cell productivity consistently and predictably in 5 L bioreactor cultivation with CRISPR-mediated targeted gene site-specific integration CHO cell line

Omics approach for generating a high-yield CHO cell line producing monoclonal antibodies

Chinese hamster ovary (CHO) cells are extensively used for the industrial manufacture of therapeutic antibodies. Generating high producing cell lines for secretory protein production requires knowing the bottleneck in the cellular machinery for protein expression. Integration site of gene of interest (GOI) is one of the important factors that influence the protein productivity. Even though screening of cells randomly integrated GOI can select high producing cells, the selected cell might not stable due to the chromosome instability. Here, we would like to look for host integration sites where GOI is high yield and stable by screening a single copy integration system. We developed several methods to identify integration sites including PCR based, whole genome sequencing based, and a platform to integrate a single copy of GOI into host genome. By determining the integration sites of the high producing clones, we can elucidate the major high yield sites for target gene expression. We have also employed the genome-editing tool, TALEN and CRISPR/cas9 to specifically integrate the vector with an antibody gene into two integration sites of CHO genome. Our data showed, IS1 and IS2 integration sites can be actively edited and specifically integrated an antibody expression vector of 15kb by either TALEN or CRISPR/Cas9. We successfully established site specifically integrated cell pools and expanded the FACS-sorted single cell into a cell line. Each single cell derived cell lines was confirmed by junction-PCR and sequence analysis. Furthermore, these single cells derived CHO cell lines are shown to express antibody gene with high titer. With the combination of omics knowledge and toolbox, including CHO genomics, transcriptomics and CHO specific microarray, GOI can be stably and highly produced

High-yield antibody production using targeted integration and engineering CHO host

To identify the high expression sites in the CHO cells, we employed NGS to analyze the integration sites of a high producing cell line (titer \u3e 3g/L). The pair-end reads with one read mapped to the vector and the other read mapped to the CHO reference genome are extracted to identify the integration sites. To test the expression activity of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed 4 integration sites are in the high producing cell line. Among the 4 integration site, one integration site was tested by CRISPR/Cas9 for target integration of antibody gene for expression. The target integrated cell pool present higher expression level (130 mg/L/copy) and less copy number when compared other integration sites. Through single-copy integration method, we can also achieve 60-150 mg/L/copy in a batch culture. About 80% of the single-copy cell clones were stable at generation 60. We have also applied the CHO-specific microarray transcriptomics technology to identify genes that contribute to high productivity. Transfection of our proprietary dual promoter vector J 1.0 resulting in 1.65 to 2.4 fold increase in the expression in engineered CHO DXB11 host. Through fed-batch process development, 3 – 5 g/L mAb productivity can be achieved through targeted integration and engineered CHO host

Development of high-producing CHO cell lines through target-designed strategy

Productivity and stability are critical for the protein drug producing cell lines for manufacturing. Given that the integration sites of gene of interest (GOI) could contribute remarkable effect on the productivity and stability of GOI expression, we intended to develop a targeting-designed approach to generate the high-producing cell lines in a time-saving and less labor-intensive method through targeting the active and stable regions. To identify the active and stable regions located in CHO genome, two approaches were applied in our experiments. Firstly, the integration sites of GOI in cell clones developed by random integration were identified by whole genome sequencing. Secondly, we developed transposon-mediated low copy integration to discover novel active region located in CHO genome. It is interesting that the productivity per integrated GOI in cell clones developed by transposon system was more than two times to that in cell clones developed by random integration (random integration: 20-40 mg/L/copy; transposon-mediated integration: 40-140mg/L/copy). In addition, about 80% of cell clones developed by transposon system maintained the stability of antibody titer after culturing for 60 generations. These results implied that the potential active and stable integration region in the cell clones developed by transposon system. The identified integration regions could be applied for target integration. In order to verify the expression activity and stability of the integration sites, we employed CRISPR/Cas9 to specifically integrate the antibody gene into CHO genome for expression. Our data showed the cell pool generated by knock-in of expression vector into the IS1 integration site present higher expression titer than cell pools generated by integration into other sites or random integration. We further cultured the single cell clones derived from this cell pool by Clonepix and limiting dilution. These single cell clones have high expression titer ranging from 254 to 804 mg/L in batch culture of after 6 Days. A single cell clone(376 mg/L in batch culture) can reached 2 g/L in fed-batch culture. The stability analysis showed this clone maintain stable expression of GOI after 60 generation. Here, we demonstrated the generation of stable cell line with high protein expression by CRISPR/Cas9 mediated target integration. This approach will cost less time and labor than traditional method

Identification of essential genes in human lymphopoiesis

Objective. Pesticide self-poisoning accounts for one-third of suicides worldwide, but few studies have investigated the national epidemiology of pesticide suicide in countries where it is a commonly used method. We investigated trends in pesticide suicide, and factors associated with such trends, in Taiwan, a rapidly developing East Asian country. Methods. We conducted an ecological study using graphical approaches and Spearman's correlation coefficients to examine trends in pesticide suicide (19872010) in Taiwan in relation to pesticide sales, bans on selected pesticides, the proportion of the workforce involved in agriculture and unemployment. We compared pesticide products banned by the Taiwanese government with products that remained on the market and pesticides that accounted for the most poisoning deaths in Taiwan. Results. Age-standardised rates of pesticide suicide showed a 67% reduction from 7.7 per 100,000 (42% of all suicides) in 1987 to 2.5 per 100,000 (12% of all suicides) in 2010, in contrast to a 69% increase in suicide rates by other methods. Pesticide poisoning was the most commonly used method of suicide in 1987 but had become the third most common method by 2010. The reduction was paralleled by a 66% fall in the workforce involved in agriculture but there was no strong evidence for its association with trends in pesticide sales, bans on selected pesticide products or unemployment. The bans mostly post-dated the decline in pesticide suicides; furthermore, they did not include products (e.g. paraquat) that accounted for most deaths and were mainly restricted to selected high-strength formulated products whilst their equivalent low-strength products were not banned. Conclusions. Access to pesticides, indicated by the size of agricultural workforce, appears to influence trends in pesticide suicide in Taiwan. Targeted bans on pesticides should focus on those products that account for most deaths. © 2012 Informa Healthcare USA, Inc.postprin