NAD+ supplementation improves mAbs productivity in CHO cells via a glucose metabolic shift

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Karyotype-based analysis of cell line instability and clonality in CHO cells

Chinese hamster ovary (CHO) cell line instability that can result in unexpected changes in phenotypes such as cell growth, productivity, or product quality is challenging for the biomanufacturing of therapeutic proteins. In addition, understanding cell line instability and its relationship to clonality is critical. We hypothesize that chromosomal rearrangements resulting from genomic instability are associated with cell line instability. We developed cell line instability models using two cell lines: secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) and their host cells (CHO-DUK). We also developed a karyotype-based framework to quantify chromosomal rearrangements. In the absence of methotrexate (MTX), long-term cultured CHO-SEAP cells exhibited a slightly increased growth rate, a significantly decreased specific productivity, and changes in the chromosomal rearrangement ratio of seven chromosomes when compared to the CHO-SEAP cells grown with MTX, demonstrating production instability. Fluorescence in situ hybridization and karyotyping analyses indicated a chromosomal loss of the SEAP gene-containing region, leading to the emergence of a non-producing subpopulation. These results support a mutation-and-selection mechanism of production instability wherein random chromosomal rearrangements can give rise to faster growing cells with low or non-producing phenotypes. Long-term cultured CHO-DUK cells exhibited an increased growth rate and an increase in the population ratio containing a particular chromosome. Growth rate and karyotyping analyses of limiting dilution CHO-DUK clones showed a correlation between the faster growing clones and the presence of that particular chromosome. Finally, karyotyping analysis indicated that CHO-DUK cells, as well as limiting dilution clones, were karyotypically heterogeneous, suggesting that chromosomal rearrangements occur spontaneously and can compromise the clonality of a cell line that has been developed from a single cell

Host cell protein control via CHO genome engineering

Chinese hamster ovary (CHO) cells, a major mammalian platform in biomanufacturing, produce and secret recombinant proteins along with host cell proteins (HCPs). Because residual HCPs in the final drug product can adversely affect (1) patients by causing immune responses, (2) drug efficacy, and (3) product stability, the effective removal of HCPs is necessary. Unfortunately, many studies have reported that many HCPs can be difficult to remove through downstream purification processes because they share similar biophysical properties to biopharmaceuticals. In this study we employed a genome engineering approach using clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR/Cas9) system-mediated knockout to address difficult-to-remove HCP problems. Three HCPs (Cathepsin D, Nidogen-1, and Prosaposin) that are known to be difficult to remove were selected, and respective knockout clones were isolated without using selective reagents or reporter genes. Clones for each HCP were characterized using various analysis methods. Taken together, we demonstrate the applicability of the CRISPR/Cas9 system to eliminate difficult-to-remove HCP expression in an industry-relevant setting

Ferromagnetic properties of (Ga,Mn)N nanowires grown by a chemical vapor deposition method

Ferromagnetic (Ga,Mn)N nanowires were grown on sapphire substrates at 900 degrees C by a chemical vapor deposition. Synchrotron radiation photoemission spectroscopy revealed that no secondary phases were found in the grown nanowire, meaning the dissolution of Mn atoms to form a solid solution in GaN nanowire. Fermi level was apart by 3.0,eV in the GaN nanowire (n-type), and it shifted toward the valance band maximum with ammonia flow rate. The Ga-to-N ratio decreased with the increase of ammonia flow rate, leading to the increase of Ga vacancies. From this, it is suggested that both increases in magnetic moment and Curie temperature with ammonia flow rate originated from the solid solution of Mn and Ga vacancies.open4

Accelerated Ulcer Healing and Resistance to Ulcer Recurrence with Gastroprotectants in Rat Model of Acetic Acid-induced Gastric Ulcer

Quality of ulcer healing (QOUH) is defined as ideal ulcer healing featuring with the fine granular ulcer scar, high functional restoration and the resistance to recurrence. This study was designed to compare the rates of QOUH achievement in rat gastric ulcer model between acid suppressant treated group and gastroprotectant treated group accompanied with elucidations of molecular mechanisms. Serosal injection of acetic acids for generating gastric ulcer and intraperitoneal (ip) injection of recombinant interleukin 1-beta (IL-1β) for recurring healed ulcer was done in SD rats. The 72 rats were divided into three groups according to treatment as follows; Group I, no further treatment, Group II, 8 weeks treatment of omeprazole, and Group III, 8 weeks of gastroprotectant treatment. IL-1β was administered for ulcer recurrence after 28 weeks of acetic acid injection. At four weeks after gastric ulcerogenesis, 58.3% (7/12) of active gastric ulcer were converted to healing stage in Group III, but 16.7% (2/12) in Group II and none in Group I, for which significant levels of epidermal growth factor, mucin, and pS2/trefoil peptide1 were contributive to these accelerated healings of Group III. ip injections of rIL-1β (200 µg/kg) at 28 weeks after acetic acid injection led to 100% of ulcer recurrence in Group I and 75.0% in Group II, but only 16.7% of Group III rats showed ulcer recurrence. Significantly attenuated levels of inflammatory cytokines including IL-2, transforming growth factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), nitrotyrosine were responsible for the resistance to ulcer recurrence in Group III. Conclusively, gastroprotectant might be prerequisite in order to achieve ideal QOUH through significant inductions of remodeling

Helicobacter pylori Infection and Halitosis – Evidence, Hypothesis, and Korean Red Ginseng to Mitigate Its Effect

Halitosis is a common and ignored condition, but in some, it is a disease-associated health problem, suggestive of overt disease conditions and hasaffected about 25–30% of world’s population, bothering nonmedical social disturbance in many people. Although two kinds, pseudohalitosis and halitophobia, are also concerned, genuine halitosis originated from the oral cavity, such as gingivitis, caries, and poor oral hygiene, in 80% and the remaining 20% are extraoral sources of halitosis, which should not be ignored because of stigmata suggestive of overt tissue dysfunctions, for instance, poor nutrition and hygiene, alcohol abuse, smoking, and systemic illness such as chronic obstructive pulmonary disease, liver cirrhosis, diabetes mellitus, and chronic renal diseases. In this chapter, Helicobacter pylori (H. pylori)–associated halitosis as one of the extragastric manifestations is introduced. Since diagnostics of halitosis includes subjective methods (examiner’s sense of smell) and objective methods (instrumental analysis), under the hypothesis of a possible relationship between H. pylori infection and objective halitosis, the real levels of volatile sulfur compounds (VSCs) in the breath showed significant correlation between VSC levels and the degree of H. pylori–associated erosive gastritis as well as gastric cancer. These findings are further validated through either measuring H2S level in gastric juices of H. pylori–infected gastritis or checking the expressions of cystathionine-γ-lyase (CSE) and cystathionine-β-synthase (CBS) responsible for H2S generation in biopsied stomach. The eradication of H. pylori significantly ameliorated halitosis, accompanied with significant reductions in gastric H2S levels (p<0.01). Korean red ginseng was very effective in either reducing H. pylori–associated H2S or alleviating halitosis in patients with H. pylori–associated chronic atrophic gastritis. Conclusively, H. pylori infection demonstrates to have an important relationship with the development of halitosis, and its eradication could possibly promote the improvement of this condition