Search Results (621)

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

Virus-like particles (VLPs), nanoscale self-assembled structures lacking viral genetic material, have emerged as a versatile platform for vaccines, targeted delivery systems, and gene-editing applications owing to their strong immunogenicity, favorable biosafety profile, and high engineerability. However, the complex architecture of VLPs, their significant size heterogeneity, and the diversity of process- and product-related impurities generated in different expression systems make downstream purification a major bottleneck limiting product quality, yield, and manufacturability. This review systematically discusses advanced downstream purification strategies for VLPs from the perspective of three major objectives: preservation of structure and biological activity, control of product heterogeneity, and assurance of viral safety. First, strategies for maintaining VLP integrity and function are examined, including optimization of solution conditions, adoption of gentle yet efficient separation operations, and integration of process analytical technology (PAT) to reduce process-induced damage. Second, the review summarizes multi-step purification approaches—spanning clarification, ultrafiltration/diafiltration (UF/DF), chromatography, and disassembly/reassembly—to remove host cell proteins, host cell DNA, and product-related impurities while improving particle homogeneity and stability. Third, viral safety is discussed primarily from the perspective of downstream virus clearance under host-dependent risk, with particular attention to orthogonal clearance steps tailored to VLP properties and expression systems such as CHO cells and insect cell–baculovirus platforms. Overall, this review provides a CQA-oriented framework and practical guidance for the development of robust, scalable, and GMP-compliant downstream purification processes for VLP-based products. 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

Metabolic dysfunction-associated fatty liver disease (MAFLD) is the most prevalent chronic liver disease worldwide with complex pathogenesis and no approved specific therapy. Siraitia grosvenorii is a widely used medicinal and edible herb, yet its efficacy and underlying mechanisms against MAFLD remain poorly defined. This study explored the protective effects and potential mechanisms of aqueous extract of Siraitia grosvenorii (AESG) on MAFLD. Based on ultra-high-performance liquid chromatography-linear trap quadrupole orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap-MS) analysis, 38 components in AESG were tentatively assigned, with tetracyclic triterpene saponins being the most abundant. In high-fat diet (HFD)-induced MAFLD mice, AESG significantly attenuated body weight gain, reduced plasma total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C) levels, and dramatically decreased hepatic triglyceride (TG) accumulation from 0.0141 mmol/g in the model group to 0.0063 mmol/g in the low-dose AESG group, corresponding to a reduction of 55.00%. AESG also alleviated plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, and improved hepatocyte steatosis. Furthermore, AESG restored HFD-induced gut dysbiosis by enriching beneficial bacteria including Akkermansia and suppressing harmful bacteria such as Ruminococcus. In free fatty acids (FFA) stimulated HepG2 cells, AESG suppressed de novo lipogenesis via downregulating Fatty Acid Synthase (FASN), Acetyl-CoA Carboxylase (ACC) and Sterol Regulatory Element-Binding Protein 1c (SREBP1c), and enhanced antioxidant capacity via activating the Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2)/Heme Oxygenase 1 (HO-1)/Sirtuin 1 (SIRT1) pathway, thereby attenuating lipid accumulation and oxidative stress. In conclusion, AESG ameliorates MAFLD by inhibiting lipogenesis, improving oxidative stress, and regulating gut microbiota. These findings support Siraitia grosvenorii as a promising natural dietary intervention for MAFLD prevention and adjuvant therapy. Full article

Botulinum neurotoxin injections are used off-label to treat chronic pain, but their efficacy is limited and paralytic effects restrict clinical utility in these applications. Here, we investigated whether combining the light chain and translocation domains of botulinum neurotoxin A (BoNT/A) with the GM1-binding B subunit of cholera toxin would be beneficial in silencing pain-associated sensory neurons. Chimeric ChoBot was assembled via a coiled-coil linking technology and was shown to retain the enzymatic activity of BoNT/A in vitro and in vivo. In cultured dorsal root ganglion neurons, ChoBot cleaved SNAP25 in a calcitonin gene-related peptide (CGRP)-rich subpopulation of sensory neurons, resulting in marked inhibition of CGRP release. ChoBot had a lesser effect on the compound muscle action potentials of the rat gastrocnemius muscle than BoNT/A following subcutaneous injections. In rat models of pain, including chemotherapy-induced peripheral neuropathy, intraplantar administration of ChoBot significantly attenuated mechanical allodynia. Immunohistochemical analysis confirmed SNAP25 cleavage in NF200- and CGRP-expressing sensory fibres in the epidermis following a single injection. ChoBot also mediated SNAP25 cleavage in human neuroblastoma cells in culture. Together, these findings indicate that ChoBot enables a silencing of pain-associated sensory pathways, providing a new strategy for the development of new long-lasting analgesics for chronic pain. Full article

Background: Respiratory syncytial virus (RSV) causes severe lung infections in infants and the elderly. The conserved central domain (CCD) of the RSV G protein is a key antigenic fragment for inducing protective antibodies. In this study, we used the hepatitis B surface antigen (HBsAg) as a platform to present this RSV G CCD fragment. Methods: We first sequenced and compared several HBsAg genotypes from clinical samples and selected one as an expression candidate for further development. The RSV G CCD was then inserted into the selected candidate to generate a recombinant expression construct. Subviral particles (SVPs) were produced using both CHO cells and yeast expression systems. Particle assembly was examined using electron microscopy. Finally, the safety and immunogenicity of the recombinant vaccine were evaluated in mice. Results: We successfully identified HBsAg38 as a potential recombinant vaccine expression candidate due to its abundant expression and secretion. The RSV G CCD fragment was inserted into the candidate and efficiently expressed in both CHO cells and yeast. The expressed protein was effectively secreted and formed uniform, spherical particles. The resulting vaccine candidate was safe for mice, causing no detectable weight loss or organ damage. Immunization with the recombinant SVPs elicited antibody responses against both HBsAg and the RSV G CCD. Upon intranasal RSV challenge, vaccinated mice exhibited markedly reduced RSV F protein and mRNA levels in lung tissues compared to PBS controls, with the yeast-derived SVP group showing the most pronounced reduction. Histopathological analysis further revealed that immunized mice had significantly less alveolar destruction and inflammatory cell infiltration than the control group, confirming that the vaccine conferred effective protection against RSV-induced lung pathology. Conclusions: We successfully developed a novel antigen-displaying HBsAg platform for generating vaccines targeting multiple pathogens. The RSV G CCD-expressing HBsAg induced a strong antibody response and provided effective protection against RSV infection. This platform offers a promising new approach for the development of next-generation vaccines. 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

Background/Objectives: Bovine ephemeral fever (BEF) is a significant disease affecting the cattle industry. The current control strategy for BEF in the field primarily relies on inactivated vaccines. However, some individuals have experienced hypersensitive reactions to these vaccines, prompting the exploration of subunit vaccines as a potential alternative for BEF prevention. Glycoprotein (G protein)-based subunit vaccines derived from virions have successfully induced neutralizing antibodies in cattle for over a decade. Nevertheless, the lack of recent studies evaluating their efficacy using recombinant proteins has raised concerns regarding the development of BEF subunit vaccines for practical field application. Therefore, the objective of this study was to evaluate the antigenicity of a novel truncated G protein produced in mammalian cells as a candidate subunit vaccine for BEF in cattle. Methods: In this study, the G protein with full ectodomain and a version truncated at the C-terminal domain were successfully generated using the ExpiCHO™ expression system. Vaccine efficacy was evaluated weekly by measuring neutralizing antibody titers and cytokine mRNA expression levels following vaccination. Results: Results show that the recombinant protein s510, derived from the G protein of BEF, can stimulate cattle to produce an average 35-fold increase in neutralizing antibodies after three doses of vaccination. The significant upregulation of IFN-γ mRNA supports the effectiveness of the s510-based subunit vaccine and indicates the activation of a cytotoxic immune response in cattle following vaccination. Conclusions: In conclusion, the results indicate that the recombinant protein s510 is a promising antigen for future BEF subunit vaccine development in this study. Full article

Background: Host cell proteins (HCPs) are process-related impurities that must be monitored in biopharmaceutical products due to their potential impact on product quality and patient safety. Targeted LC–MS/MS approaches such as multiple reaction monitoring (MRM) enable protein-specific HCP quantification but are difficult to apply in highly multiplexed assays because of retention time (RT) variability across complex bioprocess matrices. Methods: Here, we show that conventional RT-scheduled MRM workflows lack transferability when applied to heterogeneous drug substances and process intermediates. Using a targeted assay comprising 240 peptides corresponding to 97 CHO-derived HCPs, RT shifts of several minutes resulted in truncated chromatographic peaks and peptide signal loss, even when wide scheduling windows were used. To overcome this limitation, a scout-triggered MRM (st-MRM) acquisition strategy based on event-driven monitoring was implemented. Results: This approach enabled robust peptide detection across diverse matrices within a single injection, without method re-optimization. Absolute quantification using stable isotope-labeled peptides spanned six orders of magnitude, with HCPs quantified down to 2.9 ppm in purified drug substances. Conclusion: Overall, st-MRM improves the robustness and transferability of highly multiplexed targeted proteomics workflows for HCP analysis. Full article

The NerveRepack project is a European initiative that aims to develop biomimetic exoskeletons/exoprostheses for amputated or paralyzed leg patients that will receive and transmit signals to enable movements and sensations for the patient. To implement the project, it is fundamental to develop implantable neuronal electrodes that will allow bidirectional signaling between the sensors placed on the exoskeletons/exoprostheses and the nervous system. In this direction, two electrodes, plug and cuff, have been designed as integral parts of the final implantable device. The electrodes should comply with strict regulations to ensure their safe implantation in patients. The purpose of this study was to support the compliance of the implant platforms of certain key components with the ISO and ASTM standards that would be required for clinical applications. We have used an indirect method to assess the biocompatibility of the developed electrodes against neuronal cells, fibroblasts, and keratinocytes. Also, we assessed hemocompatibility, i.e., the potential of implantable electrodes to induce hemolysis or complement activation. Finally, the mutagenic/genotoxic potential was tested against the internationally recommended CHO cells. Both representative plug and cuff electrode components were found non-cytotoxic, non-mutagenic, and unable to induce hemolysis. Therefore, from the point of early evaluation of in vitro material and process biocompatibility, the selected implant platforms for the electrodes could be implanted in preclinical models to delineate their potential in vivo applications as neuronal interface with the biomimetic exoskeleton/exoprostheses. 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

Herpes simplex virus 1 (HSV-1) entry is a complex interplay of viral and host factors. The mechanisms of its regulation remain undefined. HSV-1 entry occurs via multiple distinct and cell-type dependent pathways, further complicating study of this process. HSV-1 strains with atypical entry properties aid in the elucidation of entry determinants. HSV-1 strain ANG path exhibits entry in Vero cells at 4 °C, whereas wild-type strains do not. We investigated the determinants of low temperature entry by HSV-1 ANG path in several cell types. The receptor nectin-2 mediated 4 °C entry of HSV-1 ANG path into CHO-K1 cells, but the related receptor nectin-1 did not, suggesting that gD-binding receptors are a determinant of HSV-1 entry at low temperatures. In HaCaT cells, both HSV-1 ANG path and wild-type strain KOS entered at 4 °C, while HSV-1 chimera 27/III, which contains KOS strain gB in the ANG path virus background, did not. This suggests that gB functions as a determinant of low temperature entry of HSV-1. Together, the findings suggest that there are multiple determinants and mechanisms of HSV-1 low temperature entry and that the requirements differ by cell type. Full article

Background: The ACE Y215C mutation is a common, functionally damaging missense variant (~1.5% allele frequency) associated with reduced plasma ACE levels and increased Alzheimer’s disease (AD) risk. In CHO and HEK cell models, this mutation caused a ~3–6-fold decrease in ACE surface expression, soluble ACE levels, and ACE enzymatic activity compared to those of wild-type ACE. Methods: Circulating ACE levels and activity were measured in EDTA plasma obtained from 84 carriers of the ACE Y215C mutation using a set of mAbs to the ACE. The mAbs 5B3/1G12 binding ratio was revealed as a sensitive marker for the circulating Y215C ACE mutant. Whole-exome and whole-genome sequencing (WES/WGS) were performed to identify genetic variants potentially modifying circulating ACE levels. In parallel, published sequencing and proteomic data from 35,559 Icelanders participants were analyzed to identify genes influencing ACE shedding. Sequence comparison was performed between carriers with elevated and reduced ACE concentrations to identify the potential protective variants that may compensate for decreased ACE levels due to the Y215C mutation itself. Results: Most carriers of the Y215C ACE mutation demonstrated significantly decreased ACE levels (median is 62% of control ACE levels). However, substantial inter-individual variability was observed in plasma ACE activity among carriers. Comparative sequencing analysis revealed 9648 variants unique to individuals with elevated ACE, mapping to 5779 protein-coding genes and enriched for pathways related to intracellular and transmembrane transport. Conclusions: The presence of the damaging ACE mutation Y215C does not invariably result in low plasma ACE or, likely, elevated AD risk. Therefore, combined blood ACE phenotyping and whole-exome sequencing are recommended to more accurately assess ACE-related AD susceptibility in mutation carriers. Full article