Search

Myricitrin (MYR), a flavonol consumed in the leaves and fruits of plants of the Myrtaceae family, presents anti-proliferative, anti-inflammatory, anti-diabetic, and antioxidant properties in humans. However, there are few studies regarding the cyto-genotoxicity and the chemopreventive potential of MYR. Using the in vitro Micronucleus test, the cytostasis, mutagenicity, and modulatory effect of MYR in CHO-K1 cells were assessed. The concentrations of 39 and 78 µg/mL (p < 0.001.) of MYR decrease the cytokinesis-block proliferation index (CBPI) in the short exposure treatment (4 h), while in the extended treatment (24 h), concentrations of 4.8, 9.7, 19.5, 39 and 78 µg/mL (p < 0.001.) decreased the CBPI. MYR associated with oxaliplatin decreased CBPI at all tested concentrations in the pre-(p < 0.001) and post-treatments (p < 0.001), but there was no decrease when associated with bleomycin. As for chromosome instability, MYR did not increase the frequency of micronuclei (MNi), nucleoplasmic bridges (NPBs), or nuclear buds (NBUDs) in the 4 h exposure time, however, in the 24 h treatment, MYR increased the frequency of MNi and NPBs at concentration 19.5 µg/mL (p < 0.001). As for the modulatory effect, MYR associated with bleomycin decreased the frequency of MNi, NPBs, and NBUDs at all concentrations in the pretreatment (MNi and NPBs p < 0.001, NBUDs p < 0.05) and simultaneously (MNi, NPBs and NBUDs p < 0.001). When associated with oxaliplatin, the simultaneous treatment decreased the frequency of MNi (p < 0.001) and NBUDs (p < 0.01) at all concentrations, however, in the post-treatment, MYR increased MNi (p < 0.001) and NPBs p < 0.05) in CHO-K1 cells, when compared to oxaliplatin alone. The results demonstrated that MYR could modulate the mutagenic and cytostatic actions of bleomycin and oxaliplatin, demonstrating distinct behaviors, depending on the mechanism of action of the chemotherapeutic agent. [ABSTRACT FROM AUTHOR]

The role of the FGER and GER short peptides containing a common tripeptide fragment in the regulation of the adhesive response of CHO-K1 cells was studied in this work. Both peptides increased cell adhesion both on untreated plastic and on gelatin-coated plastic, but did not affect the rate of cell attachment to poly-L-lysine-coated plastic. The GER peptide stimulated cell adhesion more on the untreated plastic. The FGER peptide increased the rate of cell attachment to gelatin over a wider range of concentrations compared to that to untreated plastic. The variativity of the process of cell spreading on different substrates under the influence of the studied peptides was noted. On untreated plastic, both peptides almost equally stimulated cell spreading. On gelatin, the FGER peptide retained the ability to stimulate cell spreading, and the GER peptide partially inhibited spreading compared to that on untreated plastic. It was found that the insertion of an additional N-terminal hydrophobic amino-acid residue Phe to the GER tripeptide fragment changes the regulatory activity of the peptide in the model of cell adhesion, depending on the stage of interaction of cells with the substrate and/or on the properties of the attachment surface. The structural and functional activity of the studied peptides with respect to various structural components of adhesive structures is discussed. [ABSTRACT FROM AUTHOR]

Introduction: Hybrid modeling combining First-Principles with machine learning is becoming a pivotal methodology for Biopharma 4.0 enactment. Chinese Hamster Ovary (CHO) cells, being the workhorse for industrial glycoproteins production, have been the object of several hybrid modeling studies. Most previous studies pursued a shallow hybrid modeling approach based on three-layered Feedforward Neural Networks (FFNNs) combined with macroscopic material balance equations. Only recently, the hybrid modeling field is incorporating deep learning into its framework with significant gains in descriptive and predictive power.Methods: This study compares, for the first time, deep and shallow hybrid modeling in a CHO process development context. Data of 24 fed-batch cultivations of a CHO-K1 cell line expressing a target glycoprotein, comprising 30 measured state variables over time, were used to compare both methodologies. Hybrid models with varying FFNN depths (3-5 layers) were systematically compared using two training methodologies. The classical training is based on the Levenberg-Marquardt algorithm, indirect sensitivity equations and cross-validation. The deep learning is based on the Adaptive Moment Estimation Method (ADAM), stochastic regularization and semidirect sensitivity equations.Results and conclusion: The results point to a systematic generalization improvement of deep hybrid models over shallow hybrid models. Overall, the training and testing errors decreased by 14.0% and 23.6% respectively when applying the deep methodology. The Central Processing Unit (CPU) time for training the deep hybrid model increased by 31.6% mainly due to the higher FFNN complexity. The final deep hybrid model is shown to predict the dynamics of the 30 state variables within the error bounds in every test experiment. Notably, the deep hybrid model could predict the metabolic shifts in key metabolites (e.g., lactate, ammonium, glutamine and glutamate) in the test experiments. We expect deep hybrid modeling to accelerate the deployment of high-fidelity digital twins in the biopharma sector in the near future.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. Key points: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing [ABSTRACT FROM AUTHOR]

In the last few years, quantum dots (QDs) have attracted research interest in different fields of science and technology. Despite their applications, it is essential to understand how nanomaterials (with different crystal sizes) are metabolized inside organisms. Thus, the focus of this study was on an evaluation of how crystal sizes of CdSTe QDs affect the viability and response of the cytochrome P450 system in CHO-K1 and HEP-G2 cells. CdSTe QDs were synthesized using a microwave-assisted system at different reaction temperatures (60, 120, 150, and 180 °C) to obtain different crystal sizes. The optical and structural characterization confirmed four crystal sizes from 3 to 8 nm. Fluorescence microscopy confirmed that CdSTe QDs are incorporated into both cell lines. Viability studies suggested that CHO-K1 cells are more sensitive than HEP-G2 cells to CdSTe QDs and Cd+2 ions. The responsible mechanisms for the toxicity of QDs and Cd+2 are apoptosis followed by necrosis. The activity of CYP 1A1, 1A2, and 3A4 isoenzymes suggests that the smallest CdSTe crystals are recognized in a manner similar to that of Cd+2. Furthermore, the largest CdSTe crystals can have different metabolic routes than Cd+2. [ABSTRACT FROM AUTHOR]

Flux balance analysis (FBA) is currently the standard method to compute metabolic fluxes in genome-scale networks. Several FBA extensions employing diverse objective functions and/or constraints have been published. Here we propose a hybrid semi-parametric FBA extension that combines mechanistic-level constraints (parametric) with empirical constraints (non-parametric) in the same linear program. A CHO dataset with 27 measured exchange fluxes obtained from 21 reactor experiments served to evaluate the method. The mechanistic constraints were deduced from a reduced CHO-K1 genome-scale network with 686 metabolites, 788 reactions and 210 degrees of freedom. The non-parametric constraints were obtained by principal component analysis of the flux dataset. The two types of constraints were integrated in the same linear program showing comparable computational cost to standard FBA. The hybrid FBA is shown to significantly improve the specific growth rate prediction under different constraints scenarios. A metabolically efficient cell growth feed targeting minimal byproducts accumulation was designed by hybrid FBA. It is concluded that integrating parametric and nonparametric constraints in the same linear program may be an efficient approach to reduce the solution space and to improve the predictive power of FBA methods when critical mechanistic information is missing. [ABSTRACT FROM AUTHOR]

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress. The screen identified both anticipated and new candidate genes that regulate ATF6α activation. Among these, calreticulin (CRT), a key ER luminal chaperone, selectively repressed ATF6α signalling: Cells lacking CRT constitutively activated a BiP::sfGFP ATF6α-dependent reporter, had higher BiP levels and an increased rate of trafficking and processing of ATF6α. Purified CRT interacted with the luminal domain of ATF6α in vitro and the two proteins co-immunoprecipitated from cell lysates. CRT depletion exposed a negative feedback loop implicating ATF6α in repressing IRE1 activity basally and overexpression of CRT reversed this repression. Our findings indicate that CRT, beyond its known role as a chaperone, also serves as an ER repressor of ATF6α to selectively regulate one arm of the UPR., Competing Interests: JT, LH, GG, HH, AO No competing interests declared, DR Reviewing editor, eLife, (© 2024, Tung et al.)

Lambda-cyhalothrin (LCT) and its microformulation Karate® (25 % a.i.) were analysed for its genotoxicity and cytotoxicity on Chinese hamster ovary (CHO-K1) cells. Cytokinesis-block micronucleus cytome (CBMN-cyt) and alkaline single-cell gel electrophoresis (SCGE) bioassays were selected to test genotoxicity. Neutral red uptake (NRU), succinic dehydrogenase activity (MTT) and apoptogenic induction were employed for estimating cytotoxicity. Both compounds were analysed within a concentration range of 0.1–100 µg/mL. Only LCT produced a significant augment in the frequency of micronuclei (MNs) when the cultures were exposed to highest concentrations of 10 and 100 µg LCT/mL. A noticeable decrease in NDI was observed for cultures treated with LCT at 10 and 100 µg/mL. Karate® induced the inhibition of both the proportion of viable cells and succinic dehydrogenase activity and triggered apoptosis 24 h of exposition. Whilst an increased GDI in CHO-K1 cells was observed in the treatments with 1–100 µg Karate®/mL, the GDI was not modified in the treatments employing LCT at equivalent doses. SCGE showed that Karate® was more prone to induce genotoxic effects than LCT. Only 50 µg/mL of Karate® was able to increase apoptosis. Our results demonstrate the genomic instability and cytotoxic effects induced by this pyrethroid insecticide, confirming that LCT exposure can result in a severe drawback for the ecological equilibrium of the environment. • Lambda-cyhalothrin (LCT) and its microparticulated product Karate® were evaluated on CHO-K1 cells. • LCT increased the frequencies of MNs and NPBs. • LCT-based Karate® microformulation induced DNA single-strand breaks. • LCT-based Karate® reduced the lysosomal and mitochondrial activities. • Only LCT-based Karate® was able to increase apoptosis after treatment for 24 h. [ABSTRACT FROM AUTHOR]

• Palmitate is not lipotoxic in granulosa cells. • Oleate induces steatosis and downregulates progesterone production in granulosa cells. • Palmitate prevents the oleate induced granulosa cell dysfunction. • cAMP levels regulate lipid droplet accumulation and progesterone production in granulosa cells. [ABSTRACT FROM AUTHOR]