Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration.

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Assessment of attenuation of varicella-zoster virus vaccines based on genomic comparison.

Live attenuated varicella-zoster virus (VZV) vaccines are used to prevent chickenpox and shingles. Single nucleotide polymorphisms (SNPs) that occur during the attenuation of parental strains are critical indicators of vaccine safety. To assess the attenuation of commercial VZV vaccines, genetic variants were comprehensively examined through high-throughput sequencing of viral DNA isolated from four VZV vaccines (Barycela, VarilRix, VariVax, and SKY Varicella). Whole-genome comparison of the four vaccines with the wild-type strain (Dumas) revealed that the sequences are highly conserved on a genome-wide scale. Among the 196 common variants across the four vaccines, 195 were already present in the genome of the parental strain (pOka), indicating that the variants occurred during the generation of the parental strain from the Dumas strain. Compared to the pOka genome, the vaccines exhibited distinct variant frequencies on a genome-wide and within an attenuation-related open reading frame. In particular, attenuation-associated 42 SNPs showed that Barycela, VarilRix, VariVax, and SKY Varicella are in ascending order regarding similarity with pOka-like genotypes, which in turn, might provide genomic evidence for the levels of attenuation. Finally, the phylogenetic network analysis demonstrated that genetic distances from the parental strain correlated with the attenuation levels of the vaccines.

α-Syntrophin alleviates ER stress to maintain protein homeostasis during myoblast differentiation.

We have previously shown evidence that α-syntrophin plays an important role in myoblast differentiation. In this study, we focused on abnormal myotube formation of the α-syntrophin knockdown C2 cell line (SNKD). The overall amount of intracellular protein and muscle-specific proteins in SNKD cells were significantly lower than those in the control. Akt-mTOR signaling, an important pathway for protein synthesis and muscle hypertrophy, was downregulated. In addition, the levels of endoplasmic reticulum (ER) stress markers increased in SNKD cells. The decrease in intracellular protein synthesis and reduction in the myotube diameter in SNKD cells were restored by 4-phenylbutyric acid, a chemical chaperone, or overexpression of α-syntrophin. These results suggest a novel role for α-syntrophin in protein homeostasis during myoblast differentiation.

Sulforaphane ameliorates serum starvation-induced muscle atrophy via activation of the Nrf2 pathway in cultured C2C12 cells.

Oxidative stress, an imbalance of redox homeostasis, contributes to the pathogenesis and progress of muscle atrophy. However, it is debated whether oxidative stress is a cause or consequence of muscle atrophy. In this study, we investigated the relationship between menadione-induced oxidative stress and serum starvation-induced muscle atrophy in C2C12 myotubes. We found that atrophic phenotypes including myotube diameter decrease, protein ubiquitination, and the expression of atrogenes were detected under oxidative stress as well as during serum starvation. Oxidative stress during serum starvation was assessed to confirm the correlation. Both intracellular reactive oxygen species (ROS) and protein oxidation were increased in atrophic myotubes. These results indicate that menadione-induced oxidative stress triggers muscle atrophy and vice versa. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key regulator of cellular response to oxidative stress and it is considered to have a cytoprotective role in the mitigation of muscle atrophy. Transcription of heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase-1, target genes of Nrf2, was decreased during serum starvation, which is related to decreased nuclear translocation of Nrf2. Pre-treatment of sulforaphane (SFN), a known Nrf2 inducer, before serum starvation showed a protective effect via Nrf2/HO-1 upregulation. SFN can liberate Nrf2 from Keap1, enabling the nuclear translocation of Nrf2. Consequently, the expression of HO-1 increased and intracellular ROS was significantly reduced by SFN pre-treatment. These results demonstrate that oxidative stress mediates the pathophysiology of muscle atrophy, which can be improved via upregulation of the Nrf2-mediated antioxidant response.

α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase.

α-Syntrophin is involved in the survival signaling pathway in myoblasts under menadione-induced oxidative stress.

Dystrophin-deficient muscle is known to be more vulnerable to oxidative stress, but not much is known about the signaling pathway(s) responsible for this phenomenon. α-Syntrophin, a component of the dystrophin-glycoprotein complex, can function as a scaffold protein because of its multiple protein interaction domains. In this study, we investigated the role of α-syntrophin in C2 myoblasts under menadione-induced oxidative stress. We found that the protein level of α-syntrophin was elevated when cells were exposed to menadione. To investigate the function of α-syntrophin during oxidative stress, we established α-syntrophin-overexpressing and knockdown cell lines. The α-syntrophin-overexpressing cells were resistant to the menadione-induced oxidative stress. In addition, survival signalings such as protein kinase B (Akt) phosphorylation and the Bcl-2/BAX ratio were increased in these cells. On the other hand, apoptotic signals such as cleavage of caspase-3 and poly ADP ribose polymerase (PARP) were increased in the α-syntrophin knockdown cells. Furthermore, Ca(2+)influx, which is known to increase when cells are exposed to oxidative stress, decreased in the α-syntrophin-overexpressing cells, but increased in the knockdown cells. These results suggest that α-syntrophin plays a pivotal role in the survival pathway triggered by menadione-induced oxidative stress in cultured myoblasts.

Triphasic mitral inflow velocity with mid-diastolic flow: the presence of mid-diastolic mitral annular velocity indicates advanced diastolic dysfunction.

Mitral inflow filling pattern usually consists of 2 forward flow velocities in sinus rhythm: early rapid filling (E) and late filling with atrial contraction (A). However, additional mid-diastolic flow velocity may be present resulting in triphasic mitral inflow filling pattern. When mitral inflow is triphasic, mitral annulus velocity recorded by tissue Doppler imaging (TDI) frequently demonstrates a mid-diastolic component (L'). The significance of L' has not been explored previously. The purpose of this study was to explore possible mechanisms and clinical implications of triphasic mitral inflow with or without L' using TDI and proBNP. Of 9004 patients who underwent transthoracic echocardiography from March to November 2003, 83 (0.9%) patients (33 male, 50 female; mean age, 63+/-10 years) with a triphasic mitral inflow velocity pattern, including mid-diastolic flow velocity of at least 0.2m/s, and sinus rhythm were prospectively identified in our clinical echocardiography laboratory. Peak velocity of E, mid-diastolic (L), and A, and deceleration time (DT) of the E wave velocity were measured. Diastolic mitral annular velocities were measured at the septal corner of the mitral annulus by TDI from the apical 4-chamber view. ProBNP was measured at the time of echocardiogram using a quantitative electrochemiluminescence immunoassay. Mean heart rate was 54+/-6 beats/min (range, 40-67). Mean left ventricular (LV) ejection fraction (EF) was 64+/-13% and LV systolic dysfunction (EF<40%) was present in only 6 (7%). Patients were classified into 2 groups: group 1 (n=47) included those who had L' and group 2 (n=36) included those without L'. Group 1 patients had significantly higher peak velocity (35+/-14 vs 26+/-6 cm/s, p=0.0002) and TVI (35+/-14 vs 26+/-6 cm/s, p=0.0002) of L, E/E' (18+/-8 vs 14+/-6, p=0.02), and left atrial volume index (42+/-14 vs 34+/-10 ml/m(2), p=0.0037). E' (4.7+/-1.3 vs 6.2+/-2.3 cm/s, p=0.001) and A' (6.2+/-2.0 vs 8.6+/-3.4 cm/s, p=0.0006) were significantly lower in group 1 compared with those of group 2. ProBNP was significantly higher in group 1 (847+/-1461 vs 438+/-1039 pmol/l, p=0.0012) and it was above normal in all except in 1 patient of group 1. In conclusion, the presence of L' in subjects with triphasic mitral inflow velocity pattern with mid-diastolic flow is associated with higher E/E', elevated proBNP and enlarged left atrium indicating advanced diastolic dysfunction with elevated filling pressures. This unique mitral annular velocity pattern should be helpful in identifying the patients with advanced diastolic dysfunction and increased LV filling pressures.