Amplification of EBNA-1 through a single-plasmid vector-based gene amplification system in HEK293 cells as an efficient transient gene expression system.

Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3'-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. KEY POINTS: • EBNA-1-amplified HEK293 cells were established using gene amplification system. • EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. • EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein.

Active Targeted Surveillance to Identify Sites of Emergence of Hantavirus.

BACKGROUND: Endemic outbreaks of hantaviruses pose a critical public health threat worldwide. Hantaan orthohantavirus (HTNV) causes hemorrhagic fever with renal syndrome (HFRS) in humans. Using comparative genomic analyses of partial and nearly complete sequences of HTNV from humans and rodents, we were able to localize, with limitations, the putative infection locations for HFRS patients. Partial sequences might not reflect precise phylogenetic positions over the whole-genome sequences; finer granularity of rodent sampling reflects more precisely the circulation of strains. METHODS: Five HFRS specimens were collected. Epidemiological surveys were conducted with the patients during hospitalization. We conducted active surveillance at suspected HFRS outbreak areas. We performed multiplex polymerase chain reaction-based next-generation sequencing to obtain the genomic sequence of HTNV from patients and rodents. The phylogeny of human- and rodent-derived HTNV was generated using the maximum likelihood method. For phylogeographic analyses, the tracing of HTNV genomes from HFRS patients was defined on the bases of epidemiological interviews, phylogenetic patterns of the viruses, and geographic locations of HTNV-positive rodents. RESULTS: The phylogeographic analyses demonstrated genetic clusters of HTNV strains from clinical specimens, with HTNV circulating in rodents at suspected sites of patient infections. CONCLUSIONS: This study demonstrates a major shift in molecular epidemiological surveillance of HTNV. Active targeted surveillance was performed at sites of suspected infections, allowing the high-resolution phylogeographic analysis to reveal the site of emergence of HTNV. We posit that this novel approach will make it possible to identify infectious sources, perform disease risk assessment, and implement preparedness against vector-borne viruses.

Multiplex PCR-Based Next-Generation Sequencing and Global Diversity of Seoul Virus in Humans and Rats.

Seoul virus (SEOV) poses a worldwide public health threat. This virus, which is harbored by Rattus norvegicus and R. rattus rats, is the causative agent of hemorrhagic fever with renal syndrome (HFRS) in humans, which has been reported in Asia, Europe, the Americas, and Africa. Defining SEOV genome sequences plays a critical role in development of preventive and therapeutic strategies against the unique worldwide hantavirus. We applied multiplex PCR-based next-generation sequencing to obtain SEOV genome sequences from clinical and reservoir host specimens. Epidemiologic surveillance of R. norvegicus rats in South Korea during 2000-2016 demonstrated that the serologic prevalence of enzootic SEOV infections was not significant on the basis of sex, weight (age), and season. Viral loads of SEOV in rats showed wide dissemination in tissues and dynamic circulation among populations. Phylogenetic analyses showed the global diversity of SEOV and possible genomic configuration of genetic exchanges.

Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells.

Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.

Role of leukotriene A4 hydrolase aminopeptidase in the pathogenesis of emphysema.

The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme with epoxy hydrolase and aminopeptidase activities. We hypothesize that the LTA4H aminopeptidase activity alleviates neutrophilic inflammation, which contributes to cigarette smoke (CS)-induced emphysema by clearing proline-glycine-proline (PGP), a triamino acid chemokine known to induce chemotaxis of neutrophils. To investigate the biological contributions made by the LTA4H aminopeptidase activity in CS-induced emphysema, we exposed wild-type mice to CS over 5 mo while treating them with a vehicle or a pharmaceutical agent (4MDM) that selectively augments the LTA4H aminopeptidase without affecting the bioproduction of leukotriene B4. Emphysematous phenotypes were assessed by premortem lung physiology with a small animal ventilator and by postmortem histologic morphometry. CS exposure acidified the airspaces and induced localization of the LTA4H protein into the nuclei of the epithelial cells. This resulted in accumulation of PGP in the airspaces by suppressing the LTA4H aminopeptidase activity. When the LTA4H aminopeptidase activity was selectively augmented by 4MDM, the levels of PGP in the bronchoalveolar lavage fluid and infiltration of neutrophils into the lungs were significantly reduced without affecting the levels of leukotriene B4. This protected murine lungs from CS-induced emphysematous alveolar remodeling. In conclusion, CS exposure promotes the development of CS-induced emphysema by suppressing the enzymatic activities of the LTA4H aminopeptidase in lung tissues and accumulating PGP and neutrophils in the airspaces. However, restoring the leukotriene A4 aminopeptidase activity with a pharmaceutical agent protected murine lungs from developing CS-induced emphysema.

Hepatitis B virus X protein overcomes all-trans retinoic acid-induced cellular senescence by downregulating levels of p16 and p21 via DNA methylation.

Despite current molecular evidence suggesting that hepatitis B virus (HBV) X protein (HBx) plays an important role during HBV-mediated hepatocarcinogenesis, the detailed mechanism is still controversial. Here, it was shown that HBx overcomes cellular senescence provoked by all-trans retinoic acid (ATRA) in HepG2 cells, as demonstrated by the impaired induction of irreversible G(1) arrest and senescence-associated ß-galactosidase activity by ATRA in the presence of HBx. The anti-senescence effect of HBx was also observed in another human hepatoma cell line, Hep3B, but not in Huh-7 cells in which the p16 and p21 proteins are absent. In addition, HBx suppressed ATRA-mediated induction of p16 and p21 in HepG2 cells via promoter hypermethylation, resulting in inactivation of retinoblastoma protein. Furthermore, the ability of HBx to overcome ATRA-induced cellular senescence almost completely disappeared when the levels of p16 and p21 in the HBx-expressing cells became similar to those in the control cells by complementation in the former by exogenous expression, knockdown of their expression in the latter using specific small interfering RNA or treatment with a DNA methylation inhibitor, 5-Aza-2'-deoxycytidine. These results suggest that HBx executes its potential by downregulating levels of p16 and p21 via DNA methylation. As cellular senescence is a tumour-suppression process, the present study provides a new strategy by which HBV promotes hepatocarcinogenesis.

All-trans retinoic acid induces cellular senescence by up-regulating levels of p16 and p21 via promoter hypomethylation.

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor ß2 whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.

A mixed herbal extract as an adjunctive therapy for attention deficit hyperactivity disorder: A randomized placebo-controlled trial.

BACKGROUND: Methylphenidate improves clinical symptoms and brain activity in attention deficit hyperactivity disorder (ADHD) patients through the attention-regulation network's dopamine system. Additionally, water-soluble extracts (HX106) of four plants (Gastrodia elata Blume, Liriope platyphylla Wang et Tang, Salvia miltiorrhiza Bunge, and Dimocarpus longan Lour) improve cognitive function. We hypothesized that the combination of HX106 and methylphenidate would improve ADHD symptoms and brain activity of the attention network more effectively than the combination of placebo and methylphenidate. METHODS: Twenty-seven patients with ADHD were administered a herbal mixture and methylphenidate (n=13), or placebo and methylphenidate (n=14) during a 4-week, randomized, double-blind, placebo-controlled clinical trial. Changes in ADHD symptoms (K-ARS scores), as well as brain activity and functional connectivity, were assessed at baseline and 4 weeks later. RESULTS: The HX106 group showed a greater improvement in total attention (16.8%) and inattention (17.2%) scores than the placebo group. The HX106 group showed increased brain activity within the left precuneus compared to the placebo group. The HX106 group also showed increased functional connectivity from the precuneus seed to the left middle temporal gyrus compared with the placebo group. In all participants, the changes in K-ARS scores were negatively correlated with changes in brain activity in the left middle temporal gyrus. CONCLUSIONS: HX106 enhanced the effect of methylphenidate on ADHD symptoms and increased brain activity in the attention-regulation network. Therefore, HX106 may be an effective adjunctive therapy for patients with ADHD.

Hepatitis B virus X protein overcomes the growth-inhibitory potential of retinoic acid by downregulating retinoic acid receptor-beta2 expression via DNA methylation.

Aberrant promoter methylation of retinoic acid receptor-beta(2) (RAR-beta(2)) is frequently detected in hepatitis B virus (HBV)-positive hepatocellular carcinoma (HCC); however, the mechanism of methylation and its biological significance are unknown. This study showed that HBx, the principal oncogene product of HBV, induced promoter hypermethylation of RAR-beta(2) via upregulation of DNA methyltransferases 1 and 3a, resulting in downregulation of its expression in human HCC cells. In addition, HBx abolished the potential of retinoic acid (RA) to downregulate levels of G(1)-checkpoint regulators including p16, p21 and p27, resulting in activation of E2F1 in the presence of RA. As a consequence, HBx-expressing cells were less susceptible to RA-induced cell growth inhibition compared with control cells. These effects almost completely disappeared when levels of RAR-beta(2) in HBx-expressing cells were restored by treatment with a universal DNA methylation inhibitor, 5-aza-2'-deoxycytidine. As RAR-beta(2) is a major executor of the anti-tumour potential of RA, its epigenetic downregulation by HBx is likely to be an important step during HBV-mediated tumorigenesis.

Differential expression of microRNAs in recombinant Chinese hamster ovary cells treated with sodium butyrate using digital RNA counting.

Sodium butyrate (NaBu) is an efficient supplement for increasing recombinant protein production in Chinese hamster ovary (CHO) cell culture. To elucidate the effects of NaBu on miRNA expression profile in recombinant CHO (rCHO) cells, differentially expressed miRNAs in NaBu-treated rCHO cells were assessed by NanoString nCounter analysis. This result showed that eight mature mouse miRNAs (let-7b, let-7d, miR-15b, miR-25, miR-27a, miR-99a, miR-125a-5p, and miR-125b-5p) were differentially expressed. Furthermore, quantitative real-time RT-PCR analysis of eight mature CHO miRNAs, annotated using a miRBase database, confirmed the transcriptomic findings. Among the potential corresponding target mRNAs for the selected mature miRNAs, seven cell growth-related target genes (e2f2, akt2, mtor, bcl-2, bim, p38α, and bmf) and five N-glycosylation-related target genes (neu1, b4galt3, gale, man1b1 and mgat4a) were selected by considering the effectiveness of NaBu on rCHO cell culture. The altered expression patterns of the 12 target mRNAs were inversely correlated with those of the selected mature miRNAs. Altogether, NanoString nCounter analysis may be useful for identifying differentially expressed miRNAs in rCHO cells.

Determination of Nicotine, Cotinine and Trans-3'-Hydroxycotinine using LC/MS/MS in Forensic Samples of a Nicotine Fatal Case by Oral Ingestion of e-cigarette Liquid.

Nicotine is a potent neurotoxin alkaloid and is used in e-cigarette liquid. The LC/MS/MS method was linear over 0.01-1.0 mg/L (r2 = 0.992-0.995). Limit of detection and limit of quantitation were 0.001 mg/L (S/N = 3) and 0.003 (S/N = 10). The inaccuracy and imprecision were <13.2%. The recoveries were >99.3%. A 39-year-old dentist was found dead lying on the floor under the couch in his dental clinic. The concentration of nicotine, cotinine, and trans-3'-hydroxycotinine (heart blood/peripheral blood) was analyzed as follows: 87.2/85.2 mg/L (ratio 1.0), 1.4/1.1 mg/L (ratio 1.3), and 0.012/0.0089 mg/L (ratio 1.3), respectively. The concentration of nicotine was determined to be 6734.8 mg/kg in gastric contents and 7262.0 mg/L in remaining e-liquid. Only, high concentration of nicotine was detected in the gastric contents as well as the two pieces of evidence collected from the death scene. This fatal case resulted from oral ingestion of e-cigarette liquid. It is estimated that at least 714 mg of nicotine was orally ingested.

Hepatitis C virus Core protein overcomes stress-induced premature senescence by down-regulating p16 expression via DNA methylation.

Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, the mechanism is still controversial. Here, we show that Core overcomes premature senescence provoked by a reactive oxygen species inducer, H2O2, in human liver cells. For this effect, Core down-regulated levels of p16 via promoter hypermethylation and subsequently induced phosphorylation of Rb in the presence of H2O2. Levels of p21 and p27, however, were little affected by Core under the condition. The potentials of Core to inactivate Rb and suppress H2O2-mediated cellular senescence were abolished when levels of p16 were recovered by either exogenous complementation or inhibition of DNA methylation. Considering that cellular senescence provoked by oxidative stresses is an important tumor suppression process, our present study provides a new strategy by which HCV promotes development of hepatocellular carcinoma.

All-trans retinoic acid induces cellular senescence via upregulation of p16, p21, and p27.

We here present a new anti-tumor mechanism of all-trans retinoic acid (ATRA). ATRA induced several biomarkers of cellular senescence including irreversible G1 arrest, morphological changes, senescence-associated ß-galactosidase, and heterochromatin foci in HepG2 cells. ATRA also upregulated levels of p16, p21, and p27 which lead to activation of Rb and subsequent inactivation of E2F1. These effects were abolished by the RNA interference-mediated silencing of p16, p21, and p27. Moreover, ATRA failed to induce cellular senescence in Huh7 and HCT116, in which p16, p21, and p27 were not upregulated by ATRA, confirming that ATRA induces cellular senescence via upregulation of p16, p21, and p27.

Hepatitis C virus Core protein stimulates cell growth by down-regulating p16 expression via DNA methylation.

Hepatitis C virus Core plays a vital role in the development of hepatocellular carcinoma; however, its action mechanism is still controversial. Here, we showed that Core down-regulated levels of p16, resulting in inactivation of Rb and subsequent activation of E2F1, which lead to growth stimulation of hepatocytes. For this effect, Core inhibited p16 expression by inducing promoter hypermethylation via up-regulation of DNA methyltransferase 1 (DNMT1) and DNMT3b. The growth stimulatory effect of Core was abolished when levels of p16 were restored by either exogenous complementation or treatment with 5-Aza-2'dC, indicating that the effect is critical for the stimulation of cell growth by Core.