Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production.

Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: • An in vitro screening system for synthetic signal peptide in mammalian cells was established • This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay • The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.

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.

Quantitative assessment of deep-seated CO2 leakage around CO2-rich springs with low soil CO2 efflux using end-member mixing analysis and carbon isotopes.

This study examined a mountainous area with two hydrochemically distinct CO2-rich springs to understand the origin, flow, and leakage of CO2, which may provide implications for precise monitoring of CO2 leakage in geological carbon storage (GCS) sites. The carbon isotopic compositions of dissolved inorganic carbon (DIC) in CO2-rich water (δ13CDIC) and those of soil CO2 (δ13CCO2) indicated a deep-seated CO2 supply to the near-surface environment in the study area. The hydrochemical difference (e.g. pH, total dissolved solids) for the two CO2-rich springs separated by 7 m, despite similar δ13CDIC and partial pressure of CO2, was considered as the result of different evolution of shallow groundwater affected by deep-seated CO2 preferentially rising along fracture zones. Electrical resistivity tomography also suggested flow through fracture zones beneath the CO2-rich springs, showing low resistivity compared to other surveyed zones. However, soil CO2 efflux was low compared to that in other natural CO2 emission sites, and in particular it was noticeably low near the CO2-rich springs, whereas δ13CCO2 was high close the CO2-rich springs. The dissolution of CO2 in the near-surface water body seemed to decrease the deep-seated CO2 leakage through the soil layer, while δ13CCO2 imprinted the source. End-member mixing analysis was performed to assess the contribution of deep-seated CO2 to the low soil CO2 efflux by assuming that atmospheric CO2 and soil CO2 (by respiration) as well as deep-seated CO2 contribute to the soil CO2 efflux. For each end-member, characteristic δ13CCO2 and CO2 concentrations were defined, and then their apportionment to soil CO2 efflux was estimated. The resultant proportion of deep-seated CO2 was up to 8.8%. Unlike the spatial distribution of high soil CO2 efflux, high proportions exceeding 3% were found around the CO2-rich springs along the east-west valley. The study results indicate that soil CO2 efflux measurement should be combined with carbon isotopic analysis in GCS sites for CO2 leakage detection because CO2 dissolution in the underground water body may blur leakage detection on the surface. The implication of this study is the need to quantitatively assess the contribution of deep-seated CO2 using the soil CO2 concentration, soil CO2 efflux, and δ13CCO2 at each measurement site.

Annual Prevalence and Incidence of Schizophrenia and Similar Psychotic Disorders in the Republic of Korea: A National Health Insurance Data-Based Study.

OBJECTIVE: We conducted this study to address the incidence and prevalence of schizophrenia and similar psychosis in South Korea with Health Insurance Review and Assessment (HIRA) database. METHODS: We used HIRA database, which includes diagnostic information of nearly all Korean nationals to collect number of cases with diagnosis of schizophrenia and schizophrenia-similar disorders (SSP), including schizophreniform, acute/transient psychotic disorders, schizoaffective disorders, and other/unspecific nonorganic psychosis (ICD-10 codes F20/23/25/28/29) between 2010 and 2015. The annual prevalence and incidence were calculated using the population data from the Korean Statistical Office. RESULTS: The 12-month prevalence of SSP of Korea between 2010 and 2015 were 0.48-0.66%. The 12-month prevalence of schizophrenia were 0.40-0.52%; The annual incidence rates (IR) of SSP between 2010 and 2015 were 118.8-148.7 per 100,000 person-year (PY). For schizophrenia, IR per 100,000 PY were 77.6-88.5 between 2010 and 2015. CONCLUSION: The 12-month prevalence found in the present study was higher than that reported in community-based epidemiologic studies in South Korea but similar to those from other countries. The annual incidence of SSP and schizophrenia was found to steadily increase and was higher than that of other countries. The high incidence rate observed in the current study needs to be studied further.