An addition of lithium chloride improves the transient gene expression yield in CHO cells

As hundreds of new recombinant therapeutic proteins enter into development each year, a cost-effective transient gene expression (TGE) system is required to assess these proteins at an early phase as a more rapid alternative to a stable gene expression system. While several strategies, such as genetic manipulation and environmental modification, have been attempted in TGE systems to improve the overall yield in Chinese hamster ovary (CHO) cells, chemical supplementation is often employed in industrial processes owing to its simplicity and effectiveness. Lithium chloride (LiCl), which is known to induce G2/M-phase cell cycle arrest, was found to be an attractive chemical additive to enhance recombinant protein production in CHO cells in a previous study by the authors. In the present study, we demonstrate that LiCl can be used as both a transfection facilitator and a production enhancer to enhance the TGE yield in CHO cells. In order to verify the effect of an addition of LiCl on transfection efficiency and episomal DNA stability in the TGE system, green fluorescent protein (GFP) was transiently transfected onto CHO cells in the absence or presence of LiCl and the fluorescence was measured by flow cytometry. At four hours post-transfection, the fluorescence signal was increased in a dose-dependent manner and was prolonged for 72 hours compared to non-added cultures, which showed weaker signals as time passed. For the TGE of monoclonal antibodies (mAb), an addition of 10mM LiCl along with DNA/PEI complexes also showed approximately a 1.3-fold increase in the final mAb concentration compared to non-added cultures. On the other hand, an addition of LiCl at various concentrations to four-hour post-transfected cultures increased the final mAb concentration to more than 3.5-fold compared to control cultures by inducing the G2/M-phase of cell cycle arrest. Taken together, the data obtained here demonstrate that LiCl is a potentially useful additive to improve TGE systems in CHO cells

Analysis of import changes through shift-share, location quotient and BCG techniques: Gwangyang Port in Asia

The main aim of this article is to analyze the import changes of Gwangyang Port using shift-share, location quotient and BCG matrix techniques. We perform the standard shift-share analysis and spatial shift-share analysis for the period 2010–2018 and investigate the import performance of Gwangyang Port for coal, iron ore, natural gas and vegetable matter. The static analysis shows that the regional shift effect, which is the most important component, is negative for coal and iron ore, but positive for natural gas and vegetable matter. The spatial shift-share analysis also indicates that Gwangyang Port experiences not only the gains in regional competitiveness but the industrial advantage for iron ore, natural gas and vegetable matter owing to its higher competitiveness. Incorporating location coefficients into BCG matrix for coal imports, we also show that Gwangyang Port succeeds upgrading its position for natural gas and vegetable matter, but fails escaping from transformation category or upgrading its position for coal and iron ore

Ribosome profiling-guided depletion of an mRNA increases cell growth rate and protein secretion

Recombinant protein production coopts the host cell machinery to provide high protein yields of industrial enzymes or biotherapeutics. However, since protein translation is energetically expensive and tightly controlled, it is unclear if highly expressed recombinant genes are translated as efficiently as host genes. Furthermore, it is unclear how the high expression impacts global translation. Here, we present the first genome-wide view of protein translation in an IgG-producing CHO cell line, measured with ribosome profiling. Through this we found that our recombinant mRNAs were translated as efficiently as the host cell transcriptome, and sequestered up to 15% of the total ribosome occupancy. During cell culture, changes in recombinant mRNA translation were consistent with changes in transcription, demonstrating that transcript levels influence specific productivity. Using this information, we identified the unnecessary resistance marker NeoR to be a highly transcribed and translated gene. Through siRNA knock-down of NeoR, we improved the production- and growth capacity of the host cell. Thus, ribosomal profiling provides valuable insights into translation in CHO cells and can guide efforts to enhance protein production

Enhancing CHO cell productivity through a novel dual selection system using Aspg and Gs in glutamine free medium

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Identification of DNA methylation changes associated with human gastric cancer

<p>Abstract</p> <p>Background</p> <p>Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult.</p> <p>Methods</p> <p>We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay) in combination with a genome analyzer and a new normalization algorithm.</p> <p>Results</p> <p>We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs), transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the <it>HOX </it>and histone gene families. We also discovered long-range epigenetic silencing (LRES) regions in gastric cancer tissue and identified several hypermethylated genes (<it>MDM2</it>, <it>DYRK2</it>, and <it>LYZ</it>) within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue.</p> <p>Conclusions</p> <p>Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.</p

Practice Pattern of Gastroenterologists for the Management of GERD Under the Minimal Influence of the Insurance Reimbursement Guideline: A Multicenter Prospective Observational Study

The objective of the study was to document practice pattern of gastroenterologists for the management of gastroesophageal reflux disease (GERD) under the minimal influence of the insurance reimbursement guideline. Data on management for 1,197 consecutive patients with typical GERD symptoms were prospectively collected during 16 weeks. In order to minimize the influence of reimbursement guideline on the use of proton pump inhibitors (PPIs), rabeprazole was used for the PPI treatment. A total of 861 patients (72%) underwent endoscopy before the start of treatment. PPIs were most commonly prescribed (87%). At the start of treatment, rabeprazole 20 mg daily was prescribed to 94% of the patients who received PPI treatment and 10 mg daily to the remaining 6%. At the third visits, rabeprazole 20 mg daily was prescribed to 70% of those who were followed and 10 mg daily for the remaining 30%. Continuous PPI treatment during the 16-week period was performed in 63% of the study patients. In conclusion, a full-dose PPI is preferred for the initial and maintenance treatment of GERD under the minimal influence of the insurance reimbursement guideline, which may reflect a high proportion of GERD patients requiring a long-term treatment of a full-dose PPI

Altered Metabolic Phenotypes and Hypothalamic Neuronal Activity Triggered by Sodium-Glucose Cotransporter 2 Inhibition

Background Sodium-glucose cotransporter 2 (SGLT-2) inhibitors are currently used to treat patients with diabetes. Previous studies have demonstrated that treatment with SGLT-2 inhibitors is accompanied by altered metabolic phenotypes. However, it has not been investigated whether the hypothalamic circuit participates in the development of the compensatory metabolic phenotypes triggered by the treatment with SGLT-2 inhibitors. Methods Mice were fed a standard diet or high-fat diet and treated with dapagliflozin, an SGLT-2 inhibitor. Food intake and energy expenditure were observed using indirect calorimetry system. The activity of hypothalamic neurons in response to dapagliflozin treatment was evaluated by immunohistochemistry with c-Fos antibody. Quantitative real-time polymerase chain reaction was performed to determine gene expression patterns in the hypothalamus of dapagliflozin-treated mice. Results Dapagliflozin-treated mice displayed enhanced food intake and reduced energy expenditure. Altered neuronal activities were observed in multiple hypothalamic nuclei in association with appetite regulation. Additionally, we found elevated immunosignals of agouti-related peptide neurons in the paraventricular nucleus of the hypothalamus. Conclusion This study suggests the functional involvement of the hypothalamus in the development of the compensatory metabolic phenotypes induced by SGLT-2 inhibitor treatment

Efficacy and safety of intragastric balloon for obesity in Korea

Background/Aims Intragastric balloon (IGB) is the only available endoscopic bariatric and metabolic therapy in Korea. End-ball (Endalis) has the longest history of clinical use among the IGBs available in Korea. However, little clinical data on this system have been reported. In this study, we aimed to evaluate the efficacy and safety of End-ball in Korea. Methods We performed a retrospective cohort study of patients who underwent IGB insertion (End-ball) from 2013 to 2019. Demographic and anthropometric data were collected. The efficacy and safety of IGB treatment were analyzed. Results In total, 80 patients were included. Mean age was 33.7 years and 83.8% were female. Initial body mass index was 34.48±4.69 kg/m2. Body mass index reduction was 3.72±2.63 kg/m2 at the time of IGB removal. Percent of total body weight loss (%TBWL) was 10.76%±6.76%. Percentage excess body weight loss was 43.67%±27.59%. Most adverse events were minor, and 71.4% of participants showed nausea, vomiting, or abdominal pain. Conclusions IGB treatment showed good efficacy and safety profile in Korean patients with obesity. In terms of %TBWL and percentage excess body weight loss, the efficacy was similar to that in the Western population