Metabolic flux analysis of CHO cells at growth and non-growth phases using isotopic tracers and mass spectrometry

Chinese hamster ovary (CHO) cells are the main platform for production of biotherapeutics in the biopharmaceutical industry. However, relatively little is known about the metabolism of CHO cells in cell culture. In this work, metabolism of CHO cells was studied at the growth phase and early stationary phase using isotopic tracers and mass spectrometry. CHO cells were grown in fed-batch culture over a period of six days. On days 2 and 4, [1,2-¹³C] glucose was introduced and the labeling of intracellular metabolites was measured by gas chromatography-mass spectrometry (GC–MS) at 6, 12 and 24 h following the introduction of tracer. Intracellular metabolic fluxes were quantified from measured extracellular rates and ¹³C-labeling dynamics of intracellular metabolites using non-stationary ¹³C-metabolic flux analysis (¹³C-MFA). The flux results revealed significant rewiring of intracellular metabolic fluxes in the transition from growth to non-growth, including changes in energy metabolism, redox metabolism, oxidative pentose phosphate pathway and anaplerosis. At the exponential phase, CHO cell metabolism was characterized by a high flux of glycolysis from glucose to lactate, anaplerosis from pyruvate to oxaloacetate and from glutamate to α-ketoglutarate, and cataplerosis though malic enzyme. At the stationary phase, the flux map was characterized by a reduced flux of glycolysis, net lactate uptake, oxidative pentose phosphate pathway flux, and reduced rate of anaplerosis. The fluxes of pyruvate dehydrogenase and TCA cycle were similar at the exponential and stationary phase. The results presented here provide a solid foundation for future studies of CHO cell metabolism for applications such as cell line development and medium optimization for high-titer production of recombinant proteins.     

Sulfated chitooligosaccharides as prolyl endopeptidase inhibitor

Prolyl endopeptidase (PEP, EC 3.4.21.26) is a proline-specific endopeptidase with a serine-type mechanism, which digests small peptide-like hormones, neuroactive peptides, and various cellular factors. PEP has been involved in neurodegenerative disorders, therefore, the discovery of PEP inhibitors can revert memory loss caused by amnesic compounds. In this study, we prepared hetero-chitooligosaccharides (COSs) with different molecular sizes using ultrafiltration (UF) membrane reactor system from hetero-chitosan with different degrees of deacetylation (DD; 90%, 75% and 50% deacetylation), and synthesized sulfated COSs (SCOSs). PEP inhibitory activities of SCOSs were evaluated and the results showed that 50% deacetylated SCOSs (50-SCOSs) exhibited higher inhibitory activities than those of 90% and 75% deacetylated SCOSs (90-SCOSs and 75-SCOSs). Among the 50-SCOSs (50-SCOS I, 5000–10,000 Da; 50-SCOS II, 1000–5000 Da; 50-SCOS III, below 1000 Da), 50-SCOS II possessed the highest inhibitory activity and IC₅₀ value was 0.38 mg/ml. Kinetics studies with 50-SCOS II indicated a competitive enzyme inhibition with a K_i value of 0.78 mg/ml. It was concluded that the 50-SCOS II may be useful for PEP inhibitor and for developing a new type PEP inhibitor from carbohydrate based materials.     

Utility of the Rio Score and Modified Rio Score in Korean Patients with Multiple Sclerosis

Objectives

Early identification of suboptimal responders to multiple sclerosis (MS) treatment is critical for optimizing therapeutic decisions. The Rio score (RS) and modified Rio score (MRS) were developed to discriminate the responses to interferon-beta (IFNB) treatment in MS patients. This study was performed to evaluate the utility of RS and MRS in daily clinical practice in Korea.

Methods

This was a real-world setting, multicenter, retrospective study of MS patients treated with IFNB from 10 hospitals in Korea. We investigated whether the RS and MRS at the early stage of IFNB therapy could predict treatment responses over 3 years. Suboptimal treatment responses at 3 years were defined as the presence of clinical relapse and/or EDSS progression and/or patients who had been treated with INFB for at least for 1 year and therapy was switched due to perceived treatment failure during the 2 years of follow-up.

Results

Seventy patients (50 females and 20 males) were enrolled; 92% (12/13) of patients with high RS and 86% (12/14) of patients with high MRS (score 2 or 3) were suboptimal responders, whereas 93% (53/57) of patients with low RS and 93% (52/56) patients with low MRS (score 0 or 1) showed optimal responses. New active lesions on MRI with clinical relapse in high RS and MRS were the most common combination in suboptimal responders.

Conclusions

We confirmed that RS and MRS at 6–15 months of IFNB therapy were useful for predicting poor responders over 3 years.     

Comparative Analysis of Disease-Linked Single Nucleotide Polymorphic Markers from Brassica rapa for Their Applicability to Brassica oleracea

Numerous studies using single nucleotide polymorphisms (SNPs) have been conducted in humans, and other animals, and in major crops, including rice, soybean, and Chinese cabbage. However, the number of SNP studies in cabbage is limited. In this present study, we evaluated whether 7,645 SNPs previously identified as molecular markers linked to disease resistance in the Brassica rapa genome could be applied to B. oleracea. In a BLAST analysis using the SNP sequences of B. rapa and B. oleracea genomic sequence data registered in the NCBI database, 256 genes for which SNPs had been identified in B. rapa were found in B. oleracea. These genes were classified into three functional groups: molecular function (64 genes), biological process (96 genes), and cellular component (96 genes). A total of 693 SNP markers, including 145 SNP markers [BRH—developed from the B. rapa genome for high-resolution melt (HRM) analysis], 425 SNP markers (BRP—based on the B. rapa genome that could be applied to B. oleracea), and 123 new SNP markers (BRS—derived from BRP and designed for HRM analysis), were investigated for their ability to amplify sequences from cabbage genomic DNA. In total, 425 of the SNP markers (BRP-based on B. rapa genome), selected from 7,645 SNPs, were successfully applied to B. oleracea. Using PCR, 108 of 145 BRH (74.5%), 415 of 425 BRP (97.6%), and 118 of 123 BRS (95.9%) showed amplification, suggesting that it is possible to apply SNP markers developed based on the B. rapa genome to B. oleracea. These results provide valuable information that can be utilized in cabbage genetics and breeding programs using molecular markers derived from other Brassica species.     

Resurrection of an alpha-1,3-galactosyltransferase gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts

Animals with a targeted disruption of genes can be produced by somatic cell nuclear transfer (SCNT). However, difficulties in clonal selection of somatic cells with a targeted mutation often result in heterogeneous nuclear donor cells, including gene-targeted and non-targeted cells, and impose a risk of producing undesired wildtype cloned animals after SCNT. In addition, the efficiency of cloning by SCNT has remained extremely low. Most cloned embryos die in utero, and the few that develop to term show a high incidence of postnatal death and abnormalities. In the present study, resurrection of an alpha-1,3-galactosyltransferase (αGT) gene-targeted miniature pig by recloning using postmortem ear skin fibroblasts was attempted. Three cloned piglets were produced from the first round of SCNT, including one stillborn and two who died immediately after birth due to respiratory distress syndrome and cardiac dysfunction. Among the three piglets, two were confirmed to be αGT gene-targeted. Fibroblasts derived from postmortem ear skin biopsies were used as nuclear donor cells for the second round of SCNT, and a piglet was produced. As expected, PCR and Southern analyses confirmed that the piglet produced from recloning was αGT gene-targeted. Currently, the piglet is fourteen months of age, and no overt health problems have been observed. Results from the present study demonstrate that loss of an invaluable animal, such as a gene-targeted miniature pig, may be rescued by recloning, with assurance of the desired genetic modification.     

Isolation of the protein tyrosine phosphatase 1B inhibitory metabolite from the marine-derived fungus Cosmospora sp. SF-5060

In the course of bioassay-guided study on the EtOAc extract of a culture broth of the marine-derived fungus Cosmospora sp. SF-5060, aquastatin A (1) was isolated as a protein tyrosine phosphatase 1B (PTP1B) inhibitory component produced by the fungus. The compound was isolated by various chromatographic methods, and the structure was determined mainly by analysis of NMR spectroscopic data. Compound 1 exhibited potent inhibitory activity against PTP1B with IC₅₀ value of 0.19 μM, and the kinetic analyses of PTP1B inhibition by compound 1 suggested that the compound is inhibiting PTP1B activity in a competitive manner. Aquastatin A (1) also showed modest but selective inhibitory activity toward PTP1B over other protein tyrosine phosphatases, such as TCPTP, SHP-2, LAR, and CD45. In addition, the result of hydrolyzing aquastatin A (1) suggested that the dihydroxypentadecyl benzoic acid moiety in the molecule is responsible for the inhibitory activity.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Effect of phosphorus deficiency on aluminium-induced citrate exudation in soybean (Glycine max)

Aluminium (Al) toxicity or phosphorus (P) deficiency can induce exudation of organic acids from the roots of some plants, which is believed to be a tolerance mechanism against Al toxicity or P deficiency. In the present study, the effect of P deficiency on Al-induced citrate exudation was investigated in three soybean varieties differing in low-P tolerance. P starvation alone failed to induce secretion of organic acids from all three soybean varieties. However, P deficiency altered Al-induced citrate exudation over time, showing a complex interaction. Short × term P starvation (4 days) produced up to 50% increase in Al-induced citrate secretion, while longer-term (10 days) starvation reduced Al-induced citrate secretion to trace amounts. However, after a further 1 day in complete nutrient solution for recovery, Al-induced citrate exudation from the recovered roots was approximately 6 times higher than that from the continuously P-starved plants, but still approximately 3.6 times lower than that from the P-sufficient control. With increasing P or Al supply, Al-induced citrate exudation increased, while Al accumulation in soybean roots decreased in parallel with the decrease of P supply. The photosynthetic rate, stomatal conductance and transpiration were decreased by P deficiency, whereas the intracellular CO₂ concentration was increased. These findings indicate that P nutrition has a significant effect on Al-induced citrate exudation and Al accumulation in soybean root apices.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.