Search Results (283)

Neonicotinoid pesticides are a typical category of emerging hazardous micropollutants, and chlorine (Cl₂) is a widely used disinfectant that readily induces the chlorination of organic micropollutants. This study systematically investigated the chlorination kinetics and transformation pathways of a representative neonicotinoid pesticide (clothianidin, CLO) and evaluated the cytotoxicity variation via Chinese Hamster Ovary (CHO) cell assays. CLO chlorination followed second-order kinetics, with a first-order dependence on both CLO and Cl₂ concentrations, and the apparent rate constant (k_app) value was measured to be 1.758 × 10⁻⁴ μM⁻¹ h⁻¹, at a pH of 7.0. The CLO chlorination initially accelerated and then retarded with the increase in pH. The same tendency was involved in the yield of disinfection byproducts (i.e., trihalomethanes and haloacetic acids). Dissolved organic matter was also a crucial factor inhibiting the chlorination of CLO. The reaction of CLO⁺ with HOCl was more prevalent than between CLO⁺ with ClO⁻, wherein HOCl likely exerts electrophilic attack either after 2-nitroguanidine hydrolysis or directly at the nitrogen sites of secondary amines. Cell exposure results revealed that the chronic cytotoxicity of CLO decreased significantly after chlorination. This study helps to the mechanistic understanding of neonicotinoid transformation during water disinfection, and provides a valuable reference for the control of neonicotinoid pesticides in drinking water. Full article

Background/Objectives: Cadherin-13 (CDH13), part of the cadherin family, is attached to the plasma membrane through glycosylphosphatidylinositol. CDH13 plays essential roles in the development of the neurological and vascular systems and is a risk factor for neural and cardiovascular diseases. CDH13 is expressed on the plasma membrane in both mature and uncleaved precursor forms with the prodomain. Although several anti-CDH13 monoclonal antibodies (mAbs) are available for basic research, there have been no reports of anti-CDH13 mAbs that can detect both the mature form and the uncleaved precursor in flow cytometry. Methods: We developed novel anti-human CDH13 mAbs (named Ca₁₃Mabs) using the mature form of CDH13-expressed cells as an antigen. Results: Among Ca₁₃Mabs, a clone, Ca₁₃Mab-17 (IgG_2b, κ) specifically recognized the mature and uncleaved precursor CDH13-overexpressed Chinese hamster ovary-K1 (CHO/CDH13) cells with no detectable cross-reactivity toward 21 other cadherins by flow cytometry. Ca₁₃Mab-17 also detected endogenous CDH13 in human glioblastoma (LN229 and U87MG) and lung mesothelioma (NCI-H2052) cell lines. The dissociation constant (K_D) value of Ca₁₃Mab-17 for LN229 was estimated at 4.1 × 10⁻⁸ M. Furthermore, Ca₁₃Mab-17 detected both the mature and uncleaved precursor CDH13 in Western blotting. It also identified new blood vessels and glioblastoma cells by immunohistochemistry. Conclusions: Ca₁₃Mab-17 is a versatile tool for detecting both mature and uncleaved precursor forms of CDH13 and has potential for tumor diagnosis and therapy. Full article

Background/Objectives: Glypican-1 (GPC1) is a heparan sulfate proteoglycan that plays a critical role in regulating various signaling pathways and tumor development. Overexpression of GPC1 promotes tumor cell proliferation and invasiveness, and is associated with poor clinical outcomes. Therefore, anti-GPC1 monoclonal antibodies (mAbs) have been developed in various modalities for tumor therapy. Methods: We developed novel anti-GPC1 mAbs using a flow cytometry-based high-throughput screening approach, the Cell-Based Immunization and Screening (CBIS) method. Results: A clone G₁Mab-28 (IgG₁, κ) reacted with GPC1-overexpressed Chinese hamster ovary-K1 (CHO/GPC1), but not parental CHO-K1, in flow cytometry. Furthermore, G₁Mab-28 recognizes the endogenous GPC1-expressing human esophageal squamous cell carcinoma KYSE770 cell line. Furthermore, G₁Mab-28 specifically recognized only CHO/GPC1, but not the other GPC family-overexpressed CHO-K1. The dissociation constant values of G₁Mab-28 for CHO/GPC1 and KYSE770 were determined to be 3.3 × 10⁻⁸ M and 4.6 × 10⁻⁹ M, respectively. Moreover, G₁Mab-28 is suitable for Western blotting and immunohistochemistry. Conclusions: G₁Mab-28, established by the CBIS method, is versatile for basic research and is expected to contribute to antibody-based tumor therapy. Full article

Leaves of Actinidia chinensis Planch. var. deliciosa (A.Chev.) A.Chev. (A. deliciosa) represent agro-industrial byproducts with potential for valorization. The present study evaluated the metabolomics profiling, cytotoxicity, genotoxicity, and antigenotoxicity of the methanolic extract of A. deliciosa leaves. The metabolomics profiling was determined using an untargeted metabolomic approach employing UPLC-HRMS. Cytotoxicity, genotoxicity, and antigenotoxicity were assessed in Chinese hamster ovary K1 (CHO-K1) cells using the in vitro cytokinesis-block micronucleus (CBMN) assay. The metabolic profile of A. deliciosa leaf extracts revealed the presence of three major classes of secondary/specialized metabolites: proanthocyanidins, flavonols, and triterpenoid saponins. Medium-polar metabolites were monomeric fla-van-3-ols, such as (+)-catechin and (−)-epicatechin, oligomeric procyanidins and prodelphinidins, and flavonols. Certain glycosylated flavonols and their derivatives, such as myricetin, quercetin, and kaempferol. Low-polarity metabolites were characterized by low-polarity triterpenoids such as maslinic, corosolic, oleanolic, and ursolic acids. At concentrations of 37.5, 75, and 150 µg/mL, the extract did not significantly increase micronuclei frequency compared to untreated control cells, indicating an absence of genotoxic potential. Moreover, co-treatment of CHO-K1 cells with the extract and mitomycin C (MMC) at 0.025 µg/mL resulted in a significant reduction in micronuclei formation induced by MMC at concentrations of 75 and 150 µg/mL, suggesting antigenotoxic activity likely associated with the phytochemical constituents presented in the extract. Full article

Hydralazine is widely used to treat hypertension during pregnancy and has epigenetic effects in cancer therapy. Cryoplatable human hepatocytes showed concentration-dependent increase in DNA damage response (linear trend p = 0.0069) following 24 h hydralazine treatment. DNA repair-deficient UV5 Chinese hamster ovary (CHO) cell lines expressing human CYP1A2 and either NAT2*4 (reference allele) or NAT2*5 (variant allele) were treated with hydralazine for 24 h. CHO cells expressing NAT2*4 showed a higher acetylation rate than those with NAT2*5 (p < 0.001), whereas CHO cell viability did not differ significantly following hydralazine treatment (p > 0.05). Hydralazine caused a concentration-dependent increase in DNA damage response in the un-transfected UV5 CHO cell line, as well as in each of the UV5 CHO cell lines transfected with human CYP1A2 and/or NAT2 alleles. CHO cells with CYP1A2 only showed higher DNA damage response from hydralazine compared to cells with CYP1A2/NAT2*4 or CYP1A2/NAT2*5 (p < 0.05 and p < 0.0001, respectively), and higher in CYP1A2/NAT2*4 versus CYP1A2/NAT2*5 cells (p = 0.0011). Apurinic/apyrimidinic (AP) sites in CHO cells expressing only CYP1A2 were significantly higher than in the un-transfected UV5 CHO cell line (p < 0.01) and higher in CHO cells expressing CYP1A2/NAT2*4 compared to CYP1A2/NAT2*5, but the difference was not significant (p > 0.05). In contrast, ROS levels were reduced following hydralazine treatment in CHO cells with CYP1A2/NAT2*4 and CYP1A2/NAT2*5 (p < 0.001 and p < 0.05, respectively). The results of the current study document DNA damage responses associated with hydralazine in human hepatocytes and CHO cells. The DNA damage response was increased following N-hydroxylation by CYP1A2, which competes with N-acetylation by NAT2. Full article

Seasonal environmental conditions can modulate the chemical composition and biological activity of essential oils from medicinal plants. This study investigated the phytochemical profile, antioxidant potential, cytotoxic activity, and cytoprotective effects of Lippia alba essential oils collected during dry and rainy seasons. Gas chromatography analysis revealed that all samples preserved a citral chemotype. Principal Component Analysis (PCA) confirmed citral as the primary discriminant metabolite, while quantitative seasonal variations were mainly associated with minor oxygenated monoterpenes, particularly geraniol, carvone, and nerolidol. The essential oil obtained during the rainy season (A5T–RS) exhibited significantly higher antioxidant activity, as determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH), reducing power, total antioxidant capacity, and hydrogen peroxide scavenging assays. Intracellular reactive oxygen species (ROS) evaluation using the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) method demonstrated that both oils reduced oxidative stress in murine fibroblasts—L929, with enhanced cytoprotective effects observed for A5T–RS. Cytotoxicity assays against non-tumor (murine fibroblast-NIH/3T3, L929, Chinese hamster ovary—CHO-K1) and tumor (human cervical carcinoma—HeLa, and human hepatocellular carcinoma—HepG2) cell lines revealed selective antiproliferative activity, with tumor cells displaying greater sensitivity, particularly to the rainy-season oil. These results demonstrate that seasonal metabolomic modulation enhances the biological performance of L. alba essential oil without altering its chemotypic identity, highlighting the importance of environmental factors in the development of bioactive plant-derived products. Full article

The type II cadherin Cadherin-19 (CDH19) plays a crucial role in neural crest development. CDH19 regulates cell–cell junctions and migration by forming catenin–cytoskeleton complexes. Although anti-CDH19 monoclonal antibodies (mAbs) are used for specific applications such as Western blotting and immunohistochemistry (IHC), suitable anti-CDH19 mAbs for flow cytometry are limited. Therefore, developing mAbs that specifically recognize cell-surface-expressed CDH19 is essential for advancing both basic research and therapeutic strategies. Here, novel anti-human CDH19 mAbs (Ca₁₉Mabs) were created using flow cytometry-based high-throughput screening. One clone, Ca₁₉Mab-8 (IgG₁, κ), specifically recognized CDH19-overexpressed Chinese hamster ovary-K1 cells but did not bind to other 21 CDHs (including both type I and type II CDHs) in flow cytometry. Additionally, Ca₁₉Mab-8 recognized endogenous CDH19 in the human glioblastoma cell line LN229. The dissociation constant (K_D) of Ca₁₉Mab-8 for LN229/CDH19 was 9.0 × 10⁻⁹ M. Ca₁₉Mab-8 also detected endogenous CDH19 in Western blotting. Furthermore, Ca₁₉Mab-8 can detect CDH19 in IHC using human melanoma tissue. These findings suggest that Ca₁₉Mab-8 is a novel mAb that detects cell-surface-expressed CDH19 with high specificity and is suitable for various applications in basic research. Therefore, Ca₁₉Mab-8 has potential for clinical diagnosis and tumor therapy. Full article

Chinese hamster ovary (CHO) cells are widely utilised in the biopharmaceutical industry to produce therapeutic proteins. Understanding the mechanisms of endoplasmic reticulum (ER) stress and its interplay with protein degradation pathways remains pivotal for improving production efficiency and product quality. In this study, we investigated the proteomic responses of CHO-K1 (non-producer), CHO DP-12 (IgG-producer), and NISTCHO (IgG-producer) cell lines under ER stress induced by a combination of the proteasome inhibitor MG132 and the glycosylation inhibitor tunicamycin. Viability, cell growth, and IgG titre were measured after 24 h, 48 h, and 72 h of treatment and the 48 h timepoint was used for the comparative analysis of the proteomic data across the three cell lines. Proteasome inhibition with MG132 intensified ER stress and altered ER-associated protein degradation (ERAD). Combined tunicamycin + MG132 treatment was associated with cell line-specific proteomic changes: NISTCHO upregulated ER translocation and glycoprotein quality control proteins (SSR4, SEC24C, UGGT1), CHO DP-12 activated redox/disulfide regulators (DNAJC10, CAPN1), while CHO-K1 showed broad proteome shifts, suggesting differences in baseline stress handling. These findings provide mechanistic insights into ER stress and protein quality control in CHO cells, offering a foundation for strategies to enhance cell line robustness and optimise biopharmaceutical production. Full article

The production of quantum dots (QDs) has increased due to their wide variety of commercial products and applications. QDs can be dangerous in the environment because their small size can encourage their incorporation into living systems. In this project, ZnS and ZnSSe were synthesized under microwave irradiation, generating a water-stable nanomaterial. The bandgap energies calculated using the UV-Vis spectra were 3.81 and 3.86 eV for ZnS and ZnSSe QDs, respectively, indicating that the selenium worked as a dopant agent. The photoluminescence analysis shows narrow emission peaks, confirming a low size distribution, and the selenium doping generated a blue shift. The crystal size of both nanomaterials was around 7 nm. The cellular toxicity of these nanomaterials was evaluated using Chinese Hamster Ovary (CHO) Cells (a standard mammalian cell model). The results suggest that ZnS and ZnSSe QDs slightly affect the viability of CHO Cells, but Zn²⁺ decreases the viability at concentrations higher than 20 mg/L. The content of zinc inside cells (by ICP-OES) suggested that QDs can enter cells more easily than Zn²⁺. Therefore, the decrease in cell viability caused by Zn²⁺ outside the cells is likely due to its effect on cell membrane integrity, suggesting that these nanomaterials are less toxic than bulk materials. Full article

Background/Objectives: Histamine H₁ receptors mediate multiple physiological and pathophysiological processes, including inflammation and allergy, by regulating downstream gene expression via transcription factors. cAMP response element-binding protein (CREB) is a major transcription factor whose phosphorylation is regulated by multiple signaling pathways. Although CREB is closely involved in multiple physiological and pathophysiological processes, the detailed intracellular signaling pathway of H₁ receptor-mediated CREB phosphorylation remains to be elucidated. We investigated the roles of G_q proteins and arrestins in H₁ receptor-mediated CREB phosphorylation. Methods: We constructed Chinese hamster ovary (CHO) expressing human wild-type (WT) H₁ receptors and two types of C-terminal mutants. One mutant was constructed by truncating the serine 487 residue only at the C-terminus (S487Trunc), and the other was constructed by substituting the serine 487 residue at the C-terminus with alanine (S487A). S487Trunc is a G_q protein-biased while S487A is an arrestin-biased receptor. The expressions of CREB and its phosphorylated form were assessed by immunoblotting. Results: Histamine promoted CREB phosphorylation in CHO cells expressing WT or S487Trunc receptors, but not in cells expressing S487A. Inhibitors of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK), and Ca²⁺ chelator suppressed histamine-induced CREB phosphorylation in CHO cells expressing WT or S487Trunc receptors. Basal CREB phosphorylation levels increased following β-arrestin overexpression and decreased after their siRNA-mediated knockdown, thus modulating histamine-stimulated CREB phosphorylation in WT CHO cells. Conclusions: H₁ receptor-mediated CREB phosphorylation is induced through G_q protein/Ca²⁺/PKC-dependent ERK and JNK activation; arrestins can modulate this process by regulating basal CREB phosphorylation. Full article

Acid-sensing ion channels (ASICs), activated under acidic conditions, play a critical role in ischemic brain injury, but the detailed mechanisms and signaling pathways remain unclear. Our previous studies have shown that activation of ASIC1a channels contributes to acidosis-induced neuronal injury, partially mediated by increased calcium influx. In this study, we provide evidence that activation of ASIC2a-containing channels induces zinc influx. In cultured mouse cortical neurons, ASIC currents that were insensitive to PcTx1 inhibition were potentiated by extracellular zinc. In Chinese Hamster Ovary cells transfected with different ASIC subunits, large inward currents were recorded upon a pH drop from 7.4 to 5.0 in cells expressing homomeric ASIC1a, ASIC2a, or heteromeric ASIC1a/2a channels when normal Na⁺-rich extracellular fluid (ECF) was used. However, when ECF was modified to one containing zinc as the primary cation, the same pH drop induced an inward current only in cells expressing homomeric ASIC2a or heteromeric ASIC1a/2a, but not homomeric ASIC1a. Fluorescence imaging revealed rapid zinc influx in cells expressing ASIC2a but not ASIC1a when zinc was applied with the acidic ECF. Additionally, at pH values where ASIC2a-containing channels were activated, acid-mediated neurotoxicity was exacerbated by zinc. Thus, ASIC2a-containing channels may represent a novel pathway for zinc entry and activation of these channels might contribute to zinc-mediated neurotoxicity. Full article

Background: Dulaglutide, a GLP-1-IgG4 Fc fusion, is a long-acting GLP-1 receptor agonist used for type 2 diabetes therapy and other emerging indications. It is produced commercially in Chinese hamster ovary (CHO) cells. The supply of the original drug is now limited in some regions, so creation of highly productive biosimilar manufacturing platforms is important. Methods: Two expression plasmids (p1.1-Tr2-Dul, p1.2-GS-Dul) encoding dulaglutide were sequentially transfected into apoptosis-resistant CHO 4BGD cells. Two-step transgene amplifications with methotrexate (MTX), followed by methionine sulfoximine (MSX) selection and subsequent cell cloning pipeline, were employed. Candidate clonal cell lines were selected using fed-batch culturing and long-term productivity testing. Results: Transfection with a second plasmid encoding glutamine synthetase (p1.2-GS-Dul) and selection with MSX resulted in a further ~30% increase titer in polyclonal population even after MTX-driven amplification. Top clone 4BGD/Dul #73 reached 1.05 g/L product titer in fed-batch culture (qP up to 22 pg·cell⁻¹·day⁻¹) and remained stable for 69 days in medium without MTX/MSX. Size exclusion-high-performance liquid chromatography showed ≥95% monomer; EC₅₀ of the purified GLP-1-Fc in a GLP-1R/CRE-Luc assay was 52 pM for the obtained product versus 76 pM for the original reference drug. Conclusions: The sequential transfection and dual-marker selection approach enables the efficient generation of a robust, high-yield, and glutamine-independent CHO producer, representing a productive strategy suitable for industrial biosimilar development. Full article

CD44 variant (CD44v) isoforms are involved in promoting cancer metastasis, sustaining cancer stem cell (CSC) properties, and conferring resistance to therapeutic interventions. Consequently, the development of monoclonal antibodies (mAbs) targeting CD44v represents a crucial strategy for eliminating CD44v-positive cancer cells. Previously, an anti-CD44v6 mAb, C₄₄Mab-9 (mouse IgG₁, κ), was established. C₄₄Mab-9 recognizes explicitly the epitope encoded by the variant exon 6-encoded region of CD44 and applies to flow cytometry, western blotting, and immunohistochemistry. To assess the therapeutic potential, a mouse IgG_2a isotype of C₄₄Mab-9 (designated C₄₄Mab-9-mG_2a) was generated, and the in vitro and in vivo antitumor activities were evaluated using gastric and colorectal cancer cell lines. C₄₄Mab-9-mG_2a demonstrated specific binding to CD44v3–10-overexpressed Chinese hamster ovary cells (CHO/CD44v3–10), as well as gastric cancer (NUGC-4) and colorectal cancer (COLO201 and COLO205) in flow cytometry. C₄₄Mab-9-mG_2a exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against CHO/CD44v3–10, NUGC-4, COLO201, and COLO205. Moreover, systemic administration of C₄₄Mab-9-mG_2a significantly inhibited tumor growth in CHO/CD44v3–10, NUGC-4, COLO201, and COLO205 xenografts compared with the control IgG_2a. These findings indicate that C₄₄Mab-9-mG_2a could be applied to the mAb-based therapy against CD44v6-positive tumors. Full article

One of the best-known flavonoid chrysin was coupled at position 7 with several trisubstituted phosphine derivatives with a flexible spacer, and their in vitro anticancer activities were investigated on 60 human tumor cell lines (NCI60) and on several gynecological cancer cells. The trisubstituted phosphines contained different substituents on the aromatic ring(s), e.g., methyl and methoxy groups or fluoro atoms. The phosphorus atom was substituted not only with aromatic rings but with cyclohexyl substituents. The ionic phosphonium building block is important because it allows the therapeutic agents to transfer across the cell membrane. Therefore, the pharmacophores linked to it can exert their effects in the mitochondria. Instead of the ionic phosphonium element, a neutral moiety, namely the triphenylmethyl group, was also added to the side chain, being sterically similar but without a charge and phosphorus atom. Most of the hybrids exhibited low micromolar growth inhibition (GI₅₀) values against the majority of the tested cell lines. Notably, conjugate 3f stood out, demonstrating nanomolar antitumor activity against the K-562 leukemia cell line (GI₅₀ = 34 nM). One selected compound (3i) with promising cancer selectivity elicited cell cycle disturbances and inhibited the migration of breast cancer. The tumor-selectivity of 3a and 3f was assessed based on their effects on non-tumor Chinese hamster ovary (CHO) cells using the CellTiter-Glo Luminescent Cell Viability Assay. Given their estimated half-maximal inhibitory concentration (IC₅₀) values on non-tumor CHO cells (2.65 µM and 1.15 µM, respectively), these conjugates demonstrate promising selectivity toward several cancer cell lines. The excellent results obtained may serve as good starting points for further optimization and the design of even more effective flavonoid- and/or phosphonium-based drugs. Full article

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology. Full article