Placental growth factor regulates the generation of TH17 cells to link angiogenesis with autoimmunity.

Helper T cells actively communicate with adjacent cells by secreting soluble mediators, yet crosstalk between helper T cells and endothelial cells remains poorly understood. Here we found that placental growth factor (PlGF), a homolog of the vascular endothelial growth factor that enhances an angiogenic switch in disease, was selectively secreted by the TH17 subset of helper T cells and promoted angiogenesis. Interestingly, the 'angio-lymphokine' PlGF, in turn, specifically induced the differentiation of pathogenic TH17 cells by activating the transcription factor STAT3 via binding to its receptors and replaced the activity of interleukin-6 in the production of interleukin-17, whereas it suppressed the generation of regulatory T cells. Moreover, T cell-derived PlGF was required for the progression of autoimmune diseases associated with TH17 differentiation, including experimental autoimmune encephalomyelitis and collagen-induced arthritis, in mice. Collectively, our findings provide insights into the PlGF-dictated links among angiogenesis, TH17 cell development and autoimmunity.

Effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoprotein in recombinant CHO cells.

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: •  The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. •  The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. •  Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.

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.

Ischemia-induced Netrin-4 promotes neovascularization through endothelial progenitor cell activation via Unc-5 Netrin receptor B.

Endothelial progenitor cells (EPCs) promote neovascularization and tissue repair by migrating to vascular injury sites; therefore, factors that enhance EPC homing to damaged tissues are of interest. Here, we provide evidence of the prominent role of the Netrin-4 (NTN4)-Unc-5 Netrin receptor B (UNC5B) axis in EPC-specific promotion of ischemic neovascularization. Our results showed that NTN4 promoted the proliferation, chemotactic migration, and paracrine effects of small EPCs (SEPCs) and significantly increased the incorporation of large EPCs (LEPCs) into tubule networks. Additionally, NTN4 prominently augmented neovascularization in mice with hindlimb ischemia by increasing the homing of exogenously transplanted EPCs to the ischemic limb and incorporating EPCs into vessels. Moreover, silencing of UNC5B, an NTN4 receptor, abrogated the NTN4-induced cellular activities of SEPCs in vitro and blood-flow recovery and neovascularization in vivo in ischemic muscle by reducing EPC homing and incorporation. These findings suggest NTN4 as an EPC-based therapy for treating angiogenesis-dependent diseases.

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.

Ginkgetin, a biflavone from Ginkgo biloba leaves, prevents adipogenesis through STAT5-mediated PPARγ and C/EBPα regulation.

Adipogenesis involved in hypertrophy and hyperplasia of adipocytes is responsible for expanding the mass of adipose tissues in obese individuals. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα) are two principal transcription factors induced by delicate signaling pathways, including signal transducer and activator of transcription 5 (STAT5), in adipogenesis. Here, we demonstrated a novel role of ginkgetin, a biflavone from Ginkgo biloba leaves, as a STAT5 inhibitor that blocks the differentiation of preadipocytes into adipocytes. During the differentiation of 3T3-L1 cells, ginkgetin treatment during the first 2 days markedly inhibited the formation of lipid-bearing adipocytes. PPARγ and C/EBPα expression was decreased in 3T3-L1 cells during adipogenesis following ginkgetin treatment, whereas no change was observed in C/EBPß or C/EBPδ expression. Inhibition of PPARγ and C/EBPα expression by ginkgetin occurred through the prevention of STAT5 activation during the initiation phase of adipogenesis. In addition, ginkgetin-mediated the inhibition of adipogenesis was recapitulated in the differentiation of primary preadipocytes. Lastly, we confirmed the inhibitory effects of ginkgetin on the hypertrophy of white adipose tissues from high-fat diet-fed mice. These results indicate that ginkgetin is a potential anti-adipogenesis and anti-obesity drug.

Two distinct cellular pathways leading to endothelial cell cytotoxicity by silica nanoparticle size.

BACKGROUND: Silica nanoparticles (SiNPs) are widely used for biosensing and diagnostics, and for the targeted delivery of therapeutic agents. Safety concerns about the biomedical and clinical applications of SiNPs have been raised, necessitating analysis of the effects of their intrinsic properties, such as sizes, shapes, and surface physicochemical characteristics, on human health to minimize risk in biomedical applications. In particular, SiNP size-associated toxicological effects, and the underlying molecular mechanisms in the vascular endothelium remain unclear. This study aimed to elucidate the detailed mechanisms underlying the cellular response to exposure to trace amounts of SiNPs and to determine applicable size criteria for biomedical application. METHODS: To clarify whether these SiNP-mediated cytotoxicity due to induction of apoptosis or necrosis, human ECs were treated with SiNPs of four different non-overlapping sizes under low serum-containing condition, stained with annexin V and propidium iodide (PI), and subjected to flow cytometric analysis (FACS). Two types of cell death mechanisms were assessed in terms of production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress induction, and autophagy activity. RESULTS: Spherical SiNPs had a diameter of 21.8 nm; this was further increased to 31.4, 42.9, and 56.7 nm. Hence, we investigated these effects in human endothelial cells (ECs) treated with these nanoparticles under overlap- or agglomerate-free conditions. The 20-nm SiNPs, but not SiNPs of other sizes, significantly induced apoptosis and necrosis. Surprisingly, the two types of cell death occurred independently and through different mechanisms. Apoptotic cell death resulted from ROS-mediated ER stress. Furthermore, autophagy-mediated necrotic cell death was induced through the PI3K/AKT/eNOS signaling axis. Together, the present results indicate that SiNPs within a diameter of < 20-nm pose greater risks to cells in terms of cytotoxic effects. CONCLUSION: These data provide novel insights into the size-dependence of the cytotoxic effects of silica nanoparticles and the underlying molecular mechanisms. The findings are expected to inform the applicable size range of SiNPs to ensure their safety in biomedical and clinical applications.

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.

Understanding of decreased sialylation of Fc-fusion protein in hyperosmotic recombinant Chinese hamster ovary cell culture: N-glycosylation gene expression and N-linked glycan antennary profile.

To understand the effects of hyperosmolality on protein glycosylation, recombinant Chinese hamster ovary (rCHO) cells producing the Fc-fusion protein were cultivated in hyperosmolar medium resulting from adding NaCl (415 mOsm/kg). The hyperosmotic culture showed increased specific Fc-fusion protein productivity (qFc ) but a decreased proportion of acidic isoforms and sialic acid content of the Fc-fusion protein. The intracellular and extracellular sialidase activities in the hyperosmotic cultures were similar to those in the control culture (314 mOsm/kg), indicating that reduced sialylation of Fc-fusion protein at hyperosmolality was not due to elevated sialidase activity. Expression of 52 N-glycosylation-related genes was assessed by the NanoString nCounter system, which provides a direct digital readout using custom-designed color-coded probes. After 3 days of hyperosmotic culture, nine genes (ugp, slc35a3, slc35d2, gcs1, manea, mgat2, mgat5b, b4galt3, and b4galt4) were differentially expressed over 1.5-fold of the control, and all these genes were down-regulated. N-linked glycan analysis by anion exchange and hydrophilic interaction HPLC showed that the proportion of highly sialylated (di-, tri-, tetra-) and tetra-antennary N-linked glycans was significantly decreased upon hyperosmotic culture. Addition of betaine, an osmoprotectant, to the hyperosmotic culture significantly increased the proportion of highly sialylated and tetra-antennary N-linked glycans (P ≤ 0.05), while it increased the expression of the N-glycan branching/antennary genes (mgat2 and mgat4b). Thus, decreased expression of the genes with roles in the N-glycan biosynthesis pathway correlated with reduced sialic acid content of Fc-fusion protein caused by hyperosmolar conditions. Taken together, the results obtained in this study provide a better understanding of the detrimental effects of hyperosmolality on N-glycosylation, especially sialylation, in rCHO cells. Biotechnol. Bioeng. 2017;114: 1721-1732. © 2017 Wiley Periodicals, Inc.

Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting.

To understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10 mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.

A single-plasmid vector for transgene amplification using short hairpin RNA targeting the 3'-UTR of amplifiable dhfr.

Gene amplification using dihydrofolate reductase gene (dhfr) and methotrexate (MTX) is widely used for recombinant protein production in mammalian cells and is typically conducted in DHFR-deficient Chinese hamster ovary (CHO) cell lines. Generation of DHFR-deficient cells can be achieved by an expression vector incorporating short hairpin RNA (shRNA) that targets the 3'-untranslated region (UTR) of endogenous dhfr. Thus, shRNAs were designed to target the 3'-UTR of endogenous dhfr, and shRNA-2 efficiently down-regulated dhfr expression in CHO-K1 cells. A single gene copy of shRNA-2 also decreased the translational level of DHFR by 80% in Flp-In CHO cells. shRNA-2 was then incorporated into a plasmid vector expressing human erythropoietin (EPO) and an exogenous DHFR to develop EPO-producing cells in the Flp-In system. The specific EPO productivity (q EPO) was enhanced by stepwise increments of MTX concentration, and differences in the amplification rate were observed in Flp-In CHO cells that expressed shRNA-2. In addition, the q EPO increased by more than 2.5-fold in the presence of 500 nM MTX. The mRNA expression level and gene copy numbers of dhfr were correlated with increased productivity in the cells, which is influenced by inhibition of endogenous dhfr. This study reveals that an expression vector including shRNA that targets the 3'-UTR of endogenous dhfr can enhance the transgene amplification rate and productivity by generating DHFR-deficient cells. This approach may be applied for amplifying the foreign gene in wild-type cell lines as a versatile single-plasmid vector.

Effect of lithium chloride on the production and sialylation of Fc-fusion protein in Chinese hamster ovary cell culture.

Lithium chloride (LiCl), which is a specific inhibitor of glycogen synthase kinase-3ß, is known to induce cell cycle arrest at the G2/M phase and to regulate apoptosis. To determine the potential of LiCl as a chemical additive to enhance specific productivity (q p) of recombinant Chinese hamster ovary (rCHO) cells through cell cycle arrest at G2/M phase, rCHO cells producing Fc-fusion protein were cultivated in serum-free media with LiCl concentrations ranging from 0 to 20 mM. The addition of LiCl induced cell cycle arrest at G2/M phase and thereby decreased the specific cell growth rate. However, LiCl increased q p in a dose-dependent manner. The beneficial effect of LiCl on q p outweighed its detrimental effect on µ, resulting in improved maximum Fc-fusion protein concentration (MFPC) at 10 mM LiCl. The q p and MFPC in the bioreactor culture with 10 mM LiCl were 5.0 and 2.1 times higher than those without LiCl, respectively. In addition, the presence of LiCl at 10 mM did not significantly affect either intracellular α2,3-ST or extracellular sialidase activity. LiCl also inhibited apoptosis of cells in the decline phase of growth by increasing Bcl-2 expression. Taken together, the results obtained in this study demonstrate the potential of LiCl as a q p-enhancing additive in CHO cell culture for improved recombinant protein production.

Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP-SETDB1 Complex for Enhancing Difficult-to-Express Protein Production.

With the emerging novel biotherapeutics that are typically difficult-to-express (DTE), improvement is required for high-yield production. To identify novel targets that can enhance DTE protein production, we performed genome-wide fluorescence-activated cell sorting (FACS)-based clustered regularly interspaced short palindromic repeats (CRISPR) knockout screening in bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cells. The screen identified the two highest-scoring genes, Atf7ip and Setdb1, which are the binding partners for H3K9me3-mediated transcriptional repression. The ATF7IP-SETDB1 complex knockout in bsAb-producing CHO cells suppressed cell growth but enhanced productivity by up to 2.7-fold. Decreased H3K9me3 levels and an increased transcriptional expression level of the transgene were also observed. Furthermore, perturbation of the ATF7IP-SETDB1 complex in monoclonal antibody (mAb)-producing CHO cells led to substantial improvements in mAb production, increasing the productivity by up to 3.9-fold without affecting the product quality. Taken together, the genome-wide FACS-based CRISPR screen identified promising targets associated with histone methylation, whose perturbation enhanced the productivity by unlocking the transgene expression.

The secreted protein Amuc_1409 from Akkermansia muciniphila improves gut health through intestinal stem cell regulation.

Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/ß-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/ß-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.

Gamma-aminobutyric acid production using immobilized glutamate decarboxylase followed by downstream processing with cation exchange chromatography.

We have developed a gamma-aminobutyric acid (GABA) production technique using his-tag mediated immobilization of Escherichia coli-derived glutamate decarboxylase (GAD), an enzyme that catalyzes the conversion of glutamate to GABA. The GAD was obtained at 1.43 g/L from GAD-overexpressed E. coli fermentation and consisted of 59.7% monomer, 29.2% dimer and 2.3% tetramer with a 97.6% soluble form of the total GAD. The harvested GAD was immobilized to metal affinity gel with an immobilization yield of 92%. Based on an investigation of specific enzyme activity and reaction characteristics, glutamic acid (GA) was chosen over monosodium glutamate (MSG) as a substrate for immobilized GAD, resulting in conversion of 2.17 M GABA in a 1 L reactor within 100 min. The immobilized enzymes retained 58.1% of their initial activities after ten consecutive uses. By using cation exchange chromatography followed by enzymatic conversion, GABA was separated from the residual substrate and leached GAD. As a consequence, the glutamic acid was mostly removed with no detectable GAD, while 91.2% of GABA was yielded in the purification step.

Streamlined in vitro screening system of synthetic signal peptides in Chinese hamster ovary cells for therapeutic protein production.

Increasing the screening efficiency and maintaining the N-terminal cleavage pattern are key factors in the development of an in vitro synthetic signal peptide screening system for high therapeutic protein production in Chinese hamster ovary (CHO) cells. This study improved the in vitro screening system of synthetic signal peptides in CHO cells for therapeutic protein production by modifying the expression vector. Incorporating a leaky stop codon with IgG transmembrane and cytoplasmic domains into the expression vector improved the proportion of high producers in establishing stable CHO cell pools. The selected signal peptides from stable CHO cell pools that were generated using degenerate codon-based oligonucleotides with a conserved polar carboxy-terminal domain in the native signal peptide showed similar N-terminal cleavage patterns to the native one. In addition, replacing native signal peptide with selected synthetic signal peptides did not influence the sialylated N-linked glycan formation and biological activity of therapeutic Fc-fusion glycoprotein in CHO cells. Thus, an in vitro synthetic signal peptide screening system can be used for therapeutic Fc-fusion glycoprotein production in CHO cells with an enhanced specific protein productivity while maintaining the N-terminal cleavage pattern similar to the native one.

Kaempferol Alleviates Mitochondrial Damage by Reducing Mitochondrial Reactive Oxygen Species Production in Lipopolysaccharide-Induced Prostate Organoids.

Common prostate diseases such as prostatitis and benign prostatic hyperplasia (BPH) have a high incidence at any age. Cellular stresses, such as reactive oxygen species (ROS) and chronic inflammation, are implicated in prostate enlargement and cancer progression and development. Kaempferol is a flavonoid found in abundance in various plants, including broccoli and spinach, and has been reported to exhibit positive biological activities, such as antioxidant and anti-inflammatory properties. In the present study, we introduced prostate organoids to investigate the protective effects of kaempferol against various cellular stresses. The levels of COX-2, iNOS, p-IκB, a pro-inflammatory cytokine, and ROS were increased by LPS treatment but reversed by kaempferol treatment. Kaempferol activated the nuclear factor erythroid 2-related factor 2(Nrf2)-related pathway and enhanced the mitochondrial quality control proteins PGC-1α, PINK1, Parkin, and Beclin. The increase in mitochondrial ROS and oxygen consumption induced by LPS was stabilized by kaempferol treatment. First, our study used prostate organoids as a novel evaluation platform. Secondly, it was demonstrated that kaempferol could alleviate the mitochondrial damage in LPS-induced induced prostate organoids by reducing the production of mitochondrial ROS.

New cell line development for antibody-producing Chinese hamster ovary cells using split green fluorescent protein.

BACKGROUND: The establishment of high producer is an important issue in Chinese hamster ovary (CHO) cell culture considering increased heterogeneity by the random integration of a transfected foreign gene and the altered position of the integrated gene. Fluorescence-activated cell sorting (FACS)-based cell line development is an efficient strategy for the selection of CHO cells in high therapeutic protein production. RESULTS: An internal ribosome entry site (IRES) was introduced for using two green fluorescence protein (GFP) fragments as a reporter to both antibody chains, the heavy chain and the light chain. The cells co-transfected with two GFP fragments showed the emission of green fluorescence by the reconstitution of split GFP. The FACS-sorted pool with GFP expression had a higher specific antibody productivity (q(Ab)) than that of the unsorted pool. The q(Ab) was highly correlated with the fluorescence intensity with a high correlation coefficient, evidenced from the analysis of median GFP and q(Ab) in individual selected clones. CONCLUSIONS: This study proved that the fragment complementation for split GFP could be an efficient indication for antibody production on the basis of high correlation of q(Ab) with reconstitution of GFP. Taken together, we developed an efficient FACS-based screening method for high antibody-producing CHO cells with the benefits of the split GFP system.

An efficient strategy for cell-based antibody library selection using an integrated vector system.

BACKGROUND: Cell panning of phage-displayed antibody library is a powerful tool for the development of therapeutic and imaging agents since disease-related cell surface proteins in native complex conformation can be directly targeted. Here, we employed a strategy taking advantage of an integrated vector system which allows rapid conversion of scFv-displaying phage into scFv-Fc format for efficient cell-based scFv library selection on a tetraspanin protein, CD9. RESULTS: A mouse scFv library constructed by using a phagemid vector, pDR-D1 was subjected to cell panning against stable CD9 transfectant, and the scFv repertoire from the enriched phage pool was directly transferred to a mammalian cassette vector, pDR-OriP-Fc1. The resulting constructs enabled transient expression of enough amounts of scFv-Fcs in HEK293E cells, and flow cytometric screening of binders for CD9 transfectant could be performed simply by using the culture supernatants. All three clones selected from the screening showed correct CD9-specificity. They could immunoprecipitate CD9 molecules out of the transfectant cell lysate and correctly stain endogenous CD9 expression on cancer cell membrane. Furthermore, competition assay with a known anti-CD9 monoclonal antibody (mAb) suggested that the binding epitopes of some of them overlap with that of the mAb which resides within the large extracellular loop of CD9. CONCLUSIONS: This study demonstrates that scFv-Fc from mammalian transient expression can be chosen as a reliable format for rapid screening and validation in cell-based scFv library selection, and the strategy described here will be applicable to efficient discovery of antibodies to diverse cell-surface targets.

Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition.

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (ß-tubulin and ß-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of ß-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.