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Sialylation during N -glycosylation plays an important role in the half-life of therapeutic glycoproteins in vivo and has sparked interest in the production of therapeutic proteins using recombinant Chinese hamster ovary (rCHO) cells. To improve the sialylation of therapeutic proteins, we examined the effect of sialyllactose supplementation on sialylation of Fc-fusion glycoproteins produced in rCHO cells. Two enzymatically-synthesized sialyllactoses, 3′-sialyllactose (3′-SL) and 6′-sialyllactose (6′-SL), were administered separately to two rCHO cell lines producing the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44, respectively. Two sialyllactoses successfully increased sialylation of Fc-fusion glycoprotein in both cell lines, as evidenced by isoform distribution, sialylated N -glycan formation, and sialic acid content. Increased sialylation by adding sialyllactose was likely the result of increased amount of intracellular CMP-sialic acid (CMP-SA), the direct nucleotide sugar for sialylation. Furthermore, the degree of sialylation enhanced by sialyllactoses was slightly effective or nearly similar compared with the addition of N -acetylmannosamine (ManNAc), a representative nucleotide sugar precursor, to increase sialylation of glycoproteins. The effectiveness of sialyllactose was also confirmed using three commercially available CHO cell culture media. Taken together, these results suggest that enzymatically-synthesized sialyllactose represents a promising candidate for culture media supplementation to increase sialylation of glycoproteins in rCHO cell culture. • Sialyllactose supplementation in rCHO cell culture significantly increased sialylation of Fc-fusion glycoproteins. • Intracellular CMP-sialic acid concentrations were significantly increased in two rCHO cells supplemented with sialyllactose. • The effect of sialyllactose on sialylation was also confirmed in three commercially available CHO cell culture media. [ABSTRACT FROM AUTHOR]

Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy. • Biomarkers characterize the physiological state of cells and help predict their behavior. • A biomarker can consist of any macromolecule present in the cell that can be detected and quantified. • Discovering and identifying biomarkers requires specific and customized methods. • In an industrial context, biomarkers enable a more rational and efficient approach to bioprocessing. • Biomarkers are often specific to environmental conditions and, as such, have limitations. [ABSTRACT FROM AUTHOR]

This investigation aimed to study the effects of adding TiO 2 on many aspects of 1393-B3 glasses, including their physio-chemical, mechanical parameters, crystallization behaviour, in-vitro bioactivity, hemocompatibility, and cellular viability. Here, we replaced 1393 glass silica with boric oxide to determine a more effective alternative. 1393-B3 bioactive glasses having the general weight % composition (53-X) B 2 O 3 , 6 Na 2 O, 5 MgO, 20 CaO, 12 K 2 O, 4 P 2 O 5 , X TiO 2 (where X = 0, 1.0, 1.5, 2.0, and 2.5 %) were prepared using solid-state melt quenching method in platinum crucible at 1100 °C for 2 h in an electric furnace with air as furnace atmosphere. This research aimed to investigate the complete substitution of 1393 glass silica with boric oxide in order to get a reduced melting temperature forming 1393-B3. The objective was to develop a more energy-efficient and economical bioactive glass. Thermal analysis of the samples was obtained up to 700 °C. To evaluate the in-vitro bioactivity, the samples were immersed in simulated body fluid (SBF). These materials undergo XRD, FTIR, and SEM/EDAX analysis both before and after soaking in SBF. These characterization results show the presence of a hydroxyapatite (HA) layer forming on the samples of the bioactive glass surface. The haemolysis process performed on human blood shows its haemocompatibility result. To test these results for the human body. We performed cell proliferation and cell compatibility on the MG-63 cell line to see its effect on osteosarcoma patients. Density (2.31–2.48 gm/cm3), pH value (7.4–8.86), and mechanical parameters such as compressive strength (98–125 MPa), flexural strength (33–54), and elasticity are measured. As a result, physio-chemical, mechanical, biological properties and in-vitro bioactivity of these samples were obtained with increasing concentration of TiO 2 in 1393-B3 borate bioactive glass and found to increase with increasing concentration of TiO 2. [ABSTRACT FROM AUTHOR]

Sesamolin, a lignan isolated from sesame oils, has been found to possess neuroprotective, anticancer, and free radical scavenging properties. We hypothesized that sesamolin could stimulate the activity of nuclear factor erythroid-derived 2-like 2 (Nrf2) and inhibit adipocyte differentiation of preadipocytes. The objective of this study was to investigate effects of sesamolin on adipocyte differentiation and its underlying molecular mechanisms. In this study, we determined the effects of treatment with 25 to 100 µM sesamolin on adipogenesis in cell culture systems. Sesamolin inhibited lipid accumulation and suppressed the expression of adipocyte markers during adipocyte differentiation of C3H10T1/2, 3T3-L1, and primary preadipocytes. Mechanism studies revealed that sesamolin increased Nrf2 protein expression without inducing its mRNA, leading to an increase in the expression of Nrf2 target genes such as heme oxygenase 1 and NAD(P)H:quinone oxidoreductase 1 (Nqo1) in C3H10T1/2 adipocytes and mouse embryonic fibroblasts. These effects were significantly attenuated in Nrf2 knockout (KO) mouse embryonic fibroblasts, indicating that effects of sesamolin were dependent on Nrf2. In H1299 human lung cancer cells with KO of Kelch like-ECH-associated protein 1 (Keap1), a negative regulator of Nrf2, sesamolin failed to further increase Nrf2 protein expression. However, upon reexpressing Keap1 in Keap1 KO cells, the ability of sesamolin to elevate Nrf2 protein expression was restored, highlighting the crucial role of Keap1 in sesamolin-induced Nrf2 activation. Taken together, these findings show that sesamolin can inhibit adipocyte differentiation through Keap1-mediated Nrf2 activation. • Sesamolin inhibits lipid accumulation during adipocyte differentiation. • Sesamolin inhibits adipocyte markers expression in C3H10T1/2 adipocytes. • Seasmolin enhances Nrf2 protein levels and its target genes expression. • The effects of sesamolin on Nrf2 activation are dependent on Keap1 protein. The effects of sesamolin on adipogenesis in C3H10T1/2 cells and molecular mechanisms were investigated in Nrf2 KO and Keap1 KO cells. Sesamolin at 50 to 100 µM activated the Nrf2, inhibited lipid accumulation and suppressed adipocyte markers expression. Sesamolin's inability to activate the Nrf2 pathway in Keap1 KO lung carcinoma cells can be rescued by reexpression of Keap1. Abbreviations: Keap1, Kelch-like ECH-associated protein 1; Nrf2, Nuclear factor erythroid-2-related factor 2; KO, knockout. [Display omitted] [ABSTRACT FROM AUTHOR]

• Nutritional aspects of the C. lanatus seeds were found as high for antioxidant activity. • Antimicrobial activity has higher affinity by the presence of various phytochemicals. • DPPH and FRAP antioxidant assays supported the radical scavenging activity. • The arrest of cell cycle of A549 cells in the G2 phase and cell viability were possessed to be greater in. • The ethanolic seed extract of C. lanatus has shown good anticancer potential against A549 cells. Cancer is a leading dangerous death-causing disease in millions over the world. It arises due to the uncontrolled growth of mal-functioned cells and formed as tumors. The disease can affect any part of the body. Still, it is a quest to find anti-cancer drugs which has been the major research endeavor around the globe. Traditionally humans use plants and their derived products as a source to cure various diseases. In recent years many researches had paved the way to use plant-based drugs. The current research aims to evaluate the anticancer activity of Citrullus lanatus seeds against A549 lung carcinoma epithelial cell lines. The study uses C. lanatus seeds which naturally contains antioxidants and phytochemicals that prevent cancer cell proliferation. The ethanolic seed extract of C. lanatus showed the presence of phytochemicals such as saponins, alkaloids, proteins and flavonoids by preliminary screening. The extract was tested for its anti-inflammatory showing 96.16 % at maximum concentration. The microbial assays also showed good signs of inhibition against the microorganisms by forming a zone of inhibition. The safety and efficacy of the extract were determined by MTT assay for the cytotoxicity using A549 cancer cell lines and non-cancerous cell lines. The IC50 concentration of C. lanatus seed extract showed dosage dependently at 51.73 μg/ml for A549 cell lines. They also resulted in significant induced cell cycle arrest and apoptosis at the G2 phase by inhibiting the A549 cell line proliferation. The present study works on lung carcinoma to find a better procurement in a traditional way that can develop future hope in the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

Vorinostat (VST) is a chemotherapeutic agent administrated for various types of cancers. However, it suffers from side effects and chemoresistance that reduce its application. Different nanoniosomes comprised Span 20, 60, 65 and 80 were prepared by the thin film hydration method and loaded with VST. The nanoniosomes were physicochemically characterized using particle size analysis and field emission scanning electron microscopy. The best formulation that was prepared using Span 65 (VST-NN-S65) included vesicle size of 127 nm with a narrow size distribution. VST-NN-S65 had an entrapment efficiency and loading capacity of 81.3 ± 5.1 and 32.0 ± 3.9 %, respectively. Drug release rate measurements showed that 90 % of VST was liberated within 1 h. Cytotoxicity assessments of VST-NN-S65 in HeLa and MCF7 cells indicated significant improvement in the effectiveness of VST, compared to the VST suspension. For VST-NN-S65, IC50 values of 26.3 and 6.6 μg mL-1 were obtained for HeLa and MCF7 cell lines, respectively. In situ apoptosis detection by the TUNEL assay revealed that apoptosis mainly occurred in the cell lines. [ABSTRACT FROM AUTHOR]

The classic way to produce single-chain (sc) glycoprotein hormones is to fuse their two subunits through the carboxy-terminal peptide (CTP) from human Choriogonadotropin (hCG). The CTP confers a longer half-life to single-chain hormones thanks to its four O-glycosyl side chains. However, unlike syncytiotrophoblastic cells, most cells used for recombinant protein production do not transfer O-glycosyl chains efficiently. We thus choose to fuse the hFSH subunits with a linker comprising two N -glycosyl side chains (sc-hFSH LNN) or none (sc-hFSH L0N), that were generated using two expression systems, HEK293 and CHO K1 cells. Their production levels and biological activities were tested and compared. Both expression systems successfully produced biologically active sc-hFSH, but, in our hands, CHO K1 cells yielded about 30-fold higher amounts of recombinant protein than HEK293 cells. Moreover, sc-hFSH L0N was considerably less expressed than sc-hFSH LNN in both cell types. Our data show that sc-hFSH L0N and sc-hFSH LNN produced from both cell lines stimulate cAMP and progesterone production in mLTC cells expressing hFSH receptors and exhibit similar B/I (in vitro Bioactivity/Immuno activity) ratios. Finally, the ratio of in vivo / in vitro bioactivities for sc-hFSH LNN relative to natural pituitary heterodimeric hFSH increased 8-fold, most likely because of a longer half-life in the blood. [ABSTRACT FROM AUTHOR]

[Display omitted] • Adult mouse hypothalamic astrocytes were immortalized to produce mHypoA-Ast1 cells. • Cell lines were also generated from cortical glia and brainstem oligodendrocytes. • The mHypoA-Ast1 cell line had a stronger response to LPS than the other two lines. • Palmitate increased Il6 and Chop in mHypoA-Ast/1 cells, but not in cortical glia. • mHypoA-Ast1 co-culture prevented Agrp induction by palmitate in mHypoE-N46 neurons. The generation of astrocyte cell lines from the hypothalamus is key to study glial involvement in hypothalamic physiology, including energy homeostasis. As such, we immortalized astrocytes from the hypothalamus of an adult male CD-1 mouse using SV40 T-antigen to generate the mHypoA-Ast1 cell line. A comparative approach was taken with two other murine GFAP-expressing cell lines that were also generated in this study: a mixed glial cell line from the cortex (mCortA-G1) and an oligodendrocyte cell line from the brainstem (mBstA-Olig1), as well as an established microglial cell line (IMG). mHypoA-Ast1 cells express GFAP, alongside other astrocytic markers such as Aldh1l1 , Aqp4 , Glt1 and S100b , and express low levels of microglial, ependymal and oligodendrocyte markers. 100 ng/mL lipopolysaccharide (LPS) elevated mRNA levels of Il6, Il1b , Tnf α and Cxcl5 in mHypoA-Ast1 cells after 4 h , while 50 μM palmitate increased Il6 and Chop mRNA, demonstrating the ability of these cells to respond to inflammatory and nutrient signals. Interestingly, co-culture of mHypoA-Ast1 cells with mHypoE-N46 hypothalamic neuronal cells prevented the palmitate-mediated increase in orexigenic neuropeptide Agrp mRNA in mHypoE-N46 cells, suggesting that this cell line can alter neuronal responses to nutrients. In conclusion, we report mHypoA-Ast1 cells representing a functional astrocyte cell line from the adult mouse brain that can be used to study the complex interactions of hypothalamic cells, as well as dysregulation that may occur in disease states, providing a key tool for neuroendocrine research. [ABSTRACT FROM AUTHOR]

The development of high-throughput anticancer drug screening methods using patient-derived cancer cell (PDC) lines that maintain their original characteristics in an in vitro three-dimensional (3D) culture system poses a significant challenge to achieving personalized cancer medicine. Because stromal tissue plays a critical role in the composition and maintenance of the cancer microenvironment, in vitro 3D-culture using reconstructed stromal tissues has attracted considerable attention. Here, a simple and unique in vitro 3D-culture method using heparin and collagen together with fibroblasts and endothelial cells to fabricate vascularized 3D-stromal tissues for in vitro culture of PDCs is reported. Whereas co-treatment with bevacizumab, a monoclonal antibody against vascular endothelial growth factor, and 5-fluorouracil significantly reduced the survival rate of 3D-cultured PDCs to 30%, separate addition of each drug did not induce comparable strong cytotoxicity, suggesting the possibility of evaluating the combined effect of anticancer drugs and angiogenesis inhibitors. Surprisingly, drug evaluation using eight PDC lines with the 3D-culture method resulted in a drug efficacy concordance rate of 75% with clinical outcomes. The model is expected to be applicable to in vitro throughput drug screening for the development of personalized cancer medicine. To replicate the cancer microenvironment, we constructed a cancer-stromal tissue model in which cancer cells are placed above and inside stromal tissue with vascular network structures derived from vascular endothelial cells in fibroblast tissue using CAViTs method. Using this method, we were able to reproduce the invasion and metastasis processes of cancer cells observed in vivo. Using patient-derived cancer cells, we assessed the possibility of evaluating the combined effect with an angiogenesis inhibitor. Further, primary cancer cells also grew on the stromal tissues with the normal medium. These data suggest that the model may be useful for new in vitro drug screening and personalized cancer medicine. [Display omitted] [ABSTRACT FROM AUTHOR]

• Free fatty acid receptors are involved in colorectal cancer pathophysiology. • Free fatty acid receptor ligands are considered promising antitumor agents. • Polyunsaturated fatty acids have proven anticancer properties. • Butyrate can support conventional therapy against colorectal cancer. Colorectal cancer (CRC) is one of the leading causes of cancer-related death. Currently, dietary factors are being emphasized in the pathogenesis of CRC. There is strong evidence that fatty acids (FAs) and free FA receptors (FFARs) are involved in CRC. This comprehensive review discusses the role of FAs and their receptors in CRC pathophysiology, development, and treatment. In particular, butyrate and n-3 polyunsaturated fatty acids have been found to exert anticancer properties by, among others, inhibiting proliferation and metastasis and inducing apoptosis in tumor cells. Consequently, they are used in conjunction with conventional therapies. Furthermore, FFAR gene expression is down-regulated in CRC, suggesting their suppressive character. Recent studies showed that the FFAR4 agonist, GW9508, can inhibit tumor growth. In conclusion, natural as well as synthetic FFAR ligands are considered promising candidates for CRC therapy. Schematic representation of the role of fatty acids on CRC development. Dietary fat components, mainly SCFAs (such as butyrate) and PUFAs, influence the risk of CRC through several mechanisms, such as inhibition of cell proliferation, maintenance of intestinal barrier, immune system modulation, epigenetic modification, and induction of apoptosis and autophagy. It is proven that free fatty acid receptors are involved in these mechanisms. Abbreviations: CRC, colorectal cancer; FFAR, free fatty acid receptor; HDACs, histone deacetylases; LCFAs, long chain fatty acids; PUFAs, polyunsaturated fatty acids; SCFAs, short chain fatty acids. [Display omitted] [ABSTRACT FROM AUTHOR]

Cultivated meat offers an avenue to feed a growing population and reduce environmental burdens associated with conventional meat production. In this Science & Society paper, we outline challenges the industry is facing in obtaining robust cell lines for the development of cultivated meat products. Through an industry survey, several knowledge gaps in cell biology were identified and are presented as research opportunities here. Continued fundamental research is essential to enhance the availability of suitable cell lines and enable cost-effective and large-scale manufacture of cultivated meat. [ABSTRACT FROM AUTHOR]

This study aimed to develop an antioxidative compound extraction method for Kratom (Mitragyna speciosa) leaves using cellulase and pectinase, in combination and alone. The enzymatic hydrolysis-assisted process (EA) successfully enhanced antioxidant extraction from kratom leaves. The single enzymatic hydrolysis-assisted process (SEA) with 6% cellulase (C6) provided the highest extraction yield. The mixed enzymatic hydrolysis-assisted process (MEA) was then developed using C6, mixed with different concentrations of pectinase. Based on the high extraction yield, the MEA using 6% cellulase and 2% pectinase (C6+P2) was selected for further studies in comparison with that from relevant SEA and the control (without EA). Different extraction processes altered the characteristics and antioxidative activities of resulting extracts. The antioxidative activity of C6+P2 was dramatically enhanced in the gastrointestinal tract model system. Fourier transform infrared (FT-IR) spectrometer results confirmed significant content of phenolic compounds, proteins, and polysaccharides in C6+P2. From in vitro cytotoxicity study, C6+P2 showed an IC 50 value of 22.86 and 4.76 µg/mL in RAW264.7 and Caco-2 cells, respectively. The bioactive compounds from C6+P2 should be identified in further studies to facilitate their application in the food and pharmaceutical industries. [Display omitted] • 6% Cellulase mixed with 2% pectinase (C6+P2) gave the highest extraction yield. • C6+P2 showed drastically increase in antioxidative activity after digestion. • FTIR spectra reconfirmed the complex form of antioxidative compounds. • Cytotoxicity of C6+P2 was evaluated as IC 50 on macrophage and epithelial cells. [ABSTRACT FROM AUTHOR]

Tissue culture techniques were used to produce large amounts of bioactive compounds with medicinal potential, overcoming space and time constraints for cancer prevention. Rice callus suspension cultures (RCSC) and seed extracts prepared from aromatic rice varieties were used to evaluate the cytotoxic impact on human colon and lung cancer cell lines, as well as a normal control cell line, using Taxol as a positive control. RCSC and seed extracts from two Indian aromatic rice varieties were applied at different concentrations to treat the cancer cell lines and normal lung fibroblasts over varying time intervals. Apoptosis was assessed in 1:5 dilutions of the A549 and HT-29 cell lines treated with RCSC for 72 h, using propidium iodide staining and flow cytometry. RCSC showed a more potent cytotoxic effect than seed extracts with minimal effect on the normal cell line, in contrast to Taxol. Confocal microscopy and flow cytometry further confirmed the apoptotic effect of RCSC. Gas chromatography-mass spectrometry-based metabolic profiling identified metabolites involved in cytotoxicity and highlighted altered pathways. RCSC is proposed as an alternative source for the development of novel anticancer drugs with reduced side effects. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells represent the most preferential host cell system for therapeutic monoclonal antibody (mAb) production. Enhancing mAb production in CHO cells can be achieved by adding chemical compounds that regulate the cell cycle and cell survival pathways. This study investigated the impact of ectoine supplementation on mAb production in CHO cells. The results showed that adding ectoine at a concentration of 100 mM on the 3rd day of cultivation improved mAb production by improving cell viability and extending the culture duration. RNA sequencing analysis revealed differentially expressed genes associated with cell cycle regulation, cell proliferation, and cellular homeostasis, in particular promotion of cell cycle arrest, which was then confirmed by flow cytometry analysis. Ectoine-treated CHO cells exhibited an increase in the number of cells in the G0/G1 phase. In addition, the cell diameter was also increased. These findings support the hypothesis that ectoine enhances mAb production in CHO cells through mechanisms involving cell cycle arrest and cellular homeostasis. Overall, this study highlights the potential of ectoine as a promising supplementation strategy to enhance mAb production not only in CHO cells but also in other cell lines. [Display omitted] • Ectoine improved mAb production in CHO cells. • Ectoine improved cell viability and extended the culture duration of CHO cells. • Ectoine increased the number of CHO cells in the G0/G1 phase. • Enhanced mAb production involved cell cycle arrest and cellular homeostasis. • Ectoine is a promising supplement to enhance mAb production in cell lines. [ABSTRACT FROM AUTHOR]

Lung cancer metastasis often leads to a poor prognosis for patients. Mesenchymal-epithelial transition (MET) is one key process associated with metastasis. MET has also been linked to multidrug drug resistance (MDR). MDR arises from the overactivity of drug efflux transporters such as P-glycoprotein (P-gp) which operate at the cell plasma membrane, under the regulatory control of the scaffold proteins ezrin (Ezr), radixin (Rdx), and moesin (Msn), collectively known as ERM proteins. The current study was intended to clarify the functional changing of P-gp and the underlying mechanisms in the context of dexamethasone (DEX)-induced MET in lung cancer cells. We found that the mRNA and membrane protein expression of Ezr and P-gp was increased in response to DEX treatment. Moreover, the DEX-treated group exhibited an increase in Rho123 efflux, and it was reversed by treatment with the P-gp inhibitor verapamil or Ezr siRNA. The decrease in cell viability with paclitaxel (PTX) treatment was mitigated by pretreatment with DEX. The increased expression and activation of P-gp during the progression of lung cancer MET was regulated by Ezr. The regulatory mechanism of P-gp expression and activity may differ depending on the cell status. [Display omitted] • EMT and MET are implicated in MDR in cancer, promoting drug efflux. • Ezr and MSN are known to be scaffold proteins for P-gp in lung cancer. • DEX-induced MET enhanced P-gp expression and activation by EZR in lung cancer. • The regulatory mechanism of P-gp expression may differ depending on the cell status. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG 1)-producing cells were generated, and the IgG 1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing. [ABSTRACT FROM AUTHOR]

Colorectal carcinoma (CRC) has become one of the most prevalent malignant tumors and exploring a potential therapeutic strategy with diminished drug-associated adverse effects to combat CRC is urgent. Herein, we designed a pH-responsive polymer to efficiently encapsulate a stimulator of interferon genes (STING) agonist (5,6- dimethylxanthenone-4-acetic acid, termed ASA404) and a common clinically used chemotherapeutic agent (1-hexylcarbamoyl-5-fluorouracil, termed HCFU). Investigations in vitro demonstrated that polymer encapsulation endowed the system with a pH-dependent disassembly behavior (pH t 6.37), which preferentially selected cancerous cells with a favorable dose reduction (dose reduction index (DRI) of HCFU was 4.09). Moreover, the growth of CRC in tumor-bearing mice was effectively suppressed, with tumor suppression rates up to 94.74%, and a combination index (CI) value of less than one (CI = 0.41 for CT26 cell lines), indicating a significant synergistic therapeutic effect. Histological analysis of the tumor micro-vessel density and enzyme-linked immunosorbent assay (ELISA) tests indicated that the system increased TNF-α and IFN-β levels in serum. Therefore, this research introduces a pH-responsive polymer-based theranostic platform with great potential for immune-chemotherapeutic and anti-vascular combination therapy of CRC. [Display omitted] [ABSTRACT FROM AUTHOR]

Molecular and cellular pathophysiological mechanisms of hypothalamus-related disorders, such as metabolic, circadian, and sleep disorders, can be studied using in vitro models of the hypothalamus. New methods to generate hypothalamic organoids are relevant for drug screening in the context of hypothalamus-related disorders. However, advances in human hypothalamic organoids are still lacking. Patient-derived induced pluripotent stem cells (iPSCs) and neurons derived from them are becoming increasingly important as experimental models for circadian rhythm disorders. Integrating data generated in vitro , in vivo , and in silico could increase the experimental data and promote new avenues of research. Multidisciplinary approaches will be necessary to achieve relevant and translational experimental models that advance our understanding of hypothalamus-related disorders and therapeutic strategies. The hypothalamus, a small and intricate brain structure, orchestrates numerous neuroendocrine functions through specialized neurons and nuclei. Disruption of this complex circuitry can result in various diseases, including metabolic, circadian, and sleep disorders. Advances in in vitro models and their integration with new technologies have significantly benefited research on hypothalamic function and pathophysiology. We explore existing in vitro hypothalamic models and address their challenges and limitations as well as translational findings. We also highlight how collaborative efforts among multidisciplinary teams are essential to develop relevant and translational experimental models capable of replicating intricate neural circuits and neuroendocrine pathways, thereby advancing our understanding of therapeutic targets and drug discovery in hypothalamus-related disorders. [ABSTRACT FROM AUTHOR]

Plants have natural compounds which possess antiproliferative potential against many cancers. In the present study, Solanum nigrum was evaluated for its anti-cancerous properties. Different compounds have been documented in literature for having significant anti-tumor and anti-cancerous activities because of the presence of biologically active compounds. Current study aimed to isolate, characterize bioactive fractions and their antiproliferative potential in human cancer cell lines. Bioactive fraction (ethyl acetate) was subjected to column chromatography for the isolation and purification of compounds responsible for cytotoxic effect followed by thin layer chromatography. NMR was performed for the identification and characterization of bioactive (SN2 and SN3) compounds. The docking analysis was performed to study the interactions of isolated compounds as a ligand with three receptors with specifically allocated PDB IDs 3NUB, 1JUH and 1F16 respectively. Two compounds (SN2 and SN3) were purified through chromatography and evaluated for antiproliferative activity. Both SN2 (Quercetin) and SN3 (Chlorogenic acid) have demonstrated anticancer activity against HepG2 cell with IC 50 value of 13.15 μg/ml and 23.63 μg/ml respectively and against HeLa cell with IC 50 value of 12.95 μg/ml and 25.50 μg/ml respectively. The results demonstrated that pure compounds isolated from S. nigrum plant exhibited anticancer activity in liver and cervical cancer cell lines along with non-toxic effect on normal cells. [Display omitted] • Bioactive compounds i.e. chlorogenic acid and Quercetin were isolated, characterized from S. nigrum. • Chlorogenic acid and Quercetin show promising cytotoxicity in human cancer cell lines. • In silico studies displayed strong binding affinity of bioactive compounds to cancer receptors. [ABSTRACT FROM AUTHOR]

[Display omitted] The acid-catalyzed reaction of 4-chloro-2,7-dimethyl- 1,8-naphthyridine with 4,6-di- tert -butyl-3-nitro-1,2-benzoquinone leads to new bis-1,3-tropolones. The crystal structures of the obtained bis-1,3-tropolones were determined by X-ray diffraction analysis; the spectral and luminescent properties, the structural and energy characteristics of the compounds were examined. The cytotoxic effect in vitro against cancer cell lines A549, H1299, HT29, CT26 and B16F10 was assessed using MTT assay. [ABSTRACT FROM AUTHOR]

Over the years, medicine has continuously tried to provide the best treatment for patients. In cancer treatment, science has been at the service of medicine, to treat and discover new and better treatment approaches to "save lives" or increase longevity. Among the new disciplines that have been used to try to meet these challenges are Big Data, Bioinformatics, Machine Learning, Data Mining, Pharmacogenomics and Genomics. Whole genome sequencing has led medicine into several fields, such as broad knowledge of the human body, high accuracy in detecting a pathology or the most promising therapeutic line for a particular individual. The aim of the study is to understand and evaluate the main similarity metrics that could eventually be used in drug recommendation systems in cancer patients. Ten of the most popular similarity indices were used in the evaluation of gene expression of twenty samples from the Genomics of Drug Sensitivity in Cancer (GDSC) Project dataset, concerning breast and skin cancer pathology. The results obtained from the tested similarity indices are discussed, proposing some of them for the mentioned types of recommendation systems. [ABSTRACT FROM AUTHOR]

Kimchi is a traditional fermented food that contains abundant nutrients and functional ingredients with various health benefits. We previously reported that kimchi active components suppress hepatic steatosis caused by endoplasmic reticulum (ER) stress in vitro and in vivo. Therefore, we assessed the effect of kimchi on the inhibition of hepatic steatosis caused by ER stress in HepG2 cells and C57BL/6N mice to verify the hypothesis that kimchi may potentially inhibit nonalcoholic fatty liver disease. We investigated the effect of kimchi on cell viability and triglyceride concentrations in cells and on lipid profile, lipid accumulation, and expression of related genes in cells and mice with hepatic steatosis. A mechanistic study was also performed using the liver X receptor α agonist T0901317 and the AMP-activated protein kinase agonist AICAR. Kimchi was noncytotoxic and effectively reduced triglyceride concentrations and suppressed hepatic steatosis-related gene expression in cells and mice. Additionally, kimchi recovered weight loss, lowered the serum and liver tissue lipid profiles, suppressed lipid accumulation, and reduced the effects of T0901317 and AICAR on lipogenic gene expression in tunicamycin-treated mice. Our results highlight that kimchi could prevent hepatic steatosis caused by ER stress in cells and mice. ER stress commonly causes hepatic steatosis in vitro and in vivo. In this study, kimchi attenuated ER stress–induced hepatic steatosis in HepG2 cells and C57BL/6N mice. Kimchi inhibited hepatic steatosis by regulating the LXRα/AMPK pathway. Abbreviations: ACC, acetyl coenzyme-A carboxylase; AMPK, AMP-activated protein kinase; ER, endoplasmic reticulum; FAS, fatty acid synthase; GOT, oxaloacetic transaminase; GPT, glutamic pyruvic transaminase; H&E, hematoxylin and eosin; ORO, oil red O; LXRα, liver X receptor α; SCD-1, stearoyl-CoA desaturase-1; SREBP-1c, sterol regulatory element-binding transcription factor 1c. [Display omitted] [ABSTRACT FROM AUTHOR]

• Most PGR combinations induced callogenesis. • Only some few PGR combinations supported callus survivor after 2 years of subcultures. • Vincristine and vinblastine are produced in homogenous-looking calluses. • Vinblastine is only produced when culturing in 2,4-D (1.1 mg/L) plus BAP 0.4 (mg/L). In this work, we evaluated the effect of 29 PGR combinations and photoperiods on callogenesis while analyzing the fate of calluses during a two-year period. Variation of PGR combinations in the culture medium strongly affected callogenesis in terms of growth and callus morphology. Although most PGR combinations resulted in callogenesis, only few ones supported cell survivor in long-term basis for the generation of cell lines as homogenous-looking calluses. Analysis by tandem liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS) revealed production of vincristine (9.25 mg/kg), vinblastine (0.17 mg/kg), and vindoline (1.02 mg/kg) in cell lines when proper combinations of PGR are employed. These callus cell lines are a promising platform to establish suspension cultures seeking production of alkaloids, particularly vincristine and vinblastine. [ABSTRACT FROM AUTHOR]

High-throughput screening (HTS) platforms have been widely used to identify candidate anticancer drugs and drug-drug combinations; however, HTS-based identification of new drug-ionizing radiation (IR) combinations has rarely been reported. Herein, we developed an integrated approach including cell-based HTS and computational large-scale isobolographic analysis to accelerate the identification of radiosensitizing compounds acting strongly and more specifically on cancer cells. In a 384-well plate format, 160 compounds likely to interfere with the cell response to radiation were screened on human glioblastoma (U251-MG) and cervix carcinoma (ME-180) cell lines, as well as on normal fibroblasts (CCD-19Lu). After drug exposure, cells were irradiated or not and short-term cell survival was assessed by high-throughput cell microscopy. Computational large-scale dose-response and isobolographic approach were used to identify promising synergistic drugs radiosensitizing cancer cells rather than normal cells. Synergy of a promising compound was confirmed on ME-180 cells by an independent 96-well assay protocol, and finally, by the gold-standard colony forming assay. We retained 4 compounds synergistic at 2 isoeffects in U251-MG and ME-180 cell lines and 11 compounds synergistically effective in only one cancer cell line. Among these 15 promising radiosensitizers, 5 compounds showed limited toxicity combined or not with IR on normal fibroblasts. Overall, this study demonstrated that HTS chemoradiation screening together with large-scale computational analysis is an efficient tool to identify synergistic drug-IR combinations, with concomitant assessment of unwanted toxicity on normal fibroblasts. It sparks expectations to accelerate the discovery of highly desired agents improving the therapeutic index of radiation therapy. [ABSTRACT FROM AUTHOR]

The application of porous bioactive ceramics as bone substitutes requires their effective osseointegration, which relies on specific physical and biological properties. In this study, we developed porous magnesium-doped hydroxyapatite (MgHA) ceramics with four different magnesium concentrations (0.25, 2, 5 and 10 mol%). The ceramics were prepared through the polymeric replication sponge method and subjected to physical and biological characterizations. Upon sintering, the porous 10 mol% MgHA ceramics exhibited increased densification, which resulted in the highest compressive strength of 2.17 MPa at the lowest porosity of 31 %. The porous samples were analysed via microcomputed tomography. The scaffolds with 0.25 and 10 mol% Mg doping concentrations were selected to elucidate the influence of Mg on morphometric parameters and biological properties at two distinct levels. The high level of Mg doping led to considerable improvements in relative bone volume, connectivity density, trabecular number and trabecular thickness. Cell attachment and proliferation tests using Vero cell lines were conducted on both samples to investigate the correlations between their morphometric parameters and bioactivities. Substantially more cells attached to and proliferated on the surface of the 10 mol% MgHA ceramics compared with those on the ceramics containing 0.25 mol% MgHA. In summary, our study underscores the effectiveness of Mg incorporation in improving the physical and biological properties of porous HA ceramics, which makes them promising candidates for bone substitute applications. [ABSTRACT FROM AUTHOR]

Autophagy is an intracellular degradative process occurring under stressful conditions. Nitric oxide (NO), a free radical, regulates autophagy and apoptosis in several cancers. However, the effect of head and neck squamous cell carcinoma (HNSCC) cell adaptation to high nitric oxide (HNO) on autophagy remains unknown. We investigated the autophagy and apoptotic changes in the HNO-adapted HNSCC cell lines. Isogenic primary HNSCC (HN18/HN30) and metastatic (HN17/HN31) cell lines were evaluated. The cells were induced with 1, 2, 3 and 4 mM DEA-NONOate, an NO donor, for 72 h and assessed for cell viability by MTT assay. "HNO-adapted cells" were defined when the cell viability in the treatment group was <10%. The surviving cells were re-treated with HNO to confirm their adaptation. HNO-adapted cells were quantified for apoptosis using flow cytometry. Autophagic structures (autophagosomes) and proteins (LC3A/B and LC3B-II) were investigated using transmission electron and confocal microscopy, respectively. HNO-adapted concentration for HN18, HN17, HN30 and HN31 cells was 3, 2, 4 and 4 mM, respectively. The HNO-adapted HN18 cells demonstrated a significantly increased apoptotic percentage, whereas no significant apoptotic change was detected in the HNO-adapted HN17, HN30 and HN31 cells compared with the parent cells. Autophagosomes were widely observed across the HNO-adapted cells. Moreover, LC3A/B and LC3B-II proteins were increased in all HNO-adapted cells. Our results demonstrate that apoptosis and/or autophagy are increased during HNO adaptation in HNSCC cell lines. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories. • Establishment and application of large-scale genome excision in CHO cells. • Targeting of non-essential genomic regions via CRISPR/Cas9 with high efficiency. • Achievement of the largest reported genomic excision of 864 kilobase pairs in CHO cells. • No negative effects on bioprocess relevant parameters after gene excision. • The concept serves as a directive towards a significantly genome reduced CHO host cell chassis. [ABSTRACT FROM AUTHOR]

The development of cisplatin resistance is one of the major causes of mammary cancer treatment failure, and is associated with changes in Sox4 gene expression. To investigate the characteristic changes that occur in canine mammary gland tumor (CMGT) cells following the development of acquired cisplatin resistance, along with the relationship between these changes and the Sox4 gene. We constructed cisplatin-resistant cell line, CHMpCIS, from the cell line CHMp, which was isolated from the primary lesion of a malignant CMGT. The biological characteristics of these cells were examined by Western blot analysis, Transwell assays, and mammosphere formation assays. Compared to CHMp cells, CHMpCIS cells exhibited elevated cisplatin resistance, apoptotic escape ability, enhanced epithelial-mesenchymal transition (EMT) and cancer stem cell (CSC) features, in addition to over-activation of the Wnt/β-catenin signaling pathway and increased Sox4 protein. In CMGT cases, CMGT tissues (CMGTT) expressed higher levels of Sox4 protein and mRNA compared to adjacent tissues (CAMGTT). We found that these changes were inhibited by silencing of Sox4 expression in CHMpCIS cells. Furthermore, activation of the Wnt/β-catenin signaling pathway increased Sox4 expression levels through a positive feedback loop. These results suggested that CHMpCIS cells circumvented the damage caused by cisplatin through altering the expression of the Sox4 gene and activating the Wnt/β-catenin pathway, thereby changing the cellular biological characteristics. • A cisplatin-resistant cell line was developed for canine mammary gland tumors. • The cell line underwent a series of alterations in its biological properties. • Sox4 gene silencing reversed altered biological properties of the cell line. • Sox4 protein and mRNA expression were increased in canine mammary gland tumor tissues. [ABSTRACT FROM AUTHOR]

Advances in the field of genomics and transcriptomics have enabled researchers to identify gene signatures related to development and treatment of Small Cell Lung Cancer. In most cases, complex gene expression patterns are identified, comprising of genes with differential behavior. Most tools use single-genes as predictors of drug response, with only limited options for multi-gene use. Here we examine the potential of predicting drug response using these complex gene expression signatures by employing clustering and signal enrichment in Small Cell Lung Cancer. Our results demonstrate clustering genes from complex expression patterns helps identify differential activity of gene groups with alternate function which can then be used to predict drug response. • Clustering genes in expression signatures before enrichment analysis helps find key groups • Most small cell lung cancer cell lines are enriched for epithelial genes • A subset of inflamed signature genes used to predict immunotherapy response are not expressed in cell lines [ABSTRACT FROM AUTHOR]

Sesamin and sesamolin are major sesame lignans that have demonstrated anti-inflammatory, anticancer, and neuroprotective properties and potential benefits in the liver, cardiovascular diseases, and metabolic syndrome. However, despite previous research on their antiobesity effects and underlying mechanisms, a comprehensive investigation of these aspects is still lacking. In this study, we evaluated the regulatory effects of 20 to 80 µM sesamin and sesamolin on adipogenesis in vitro using 3T3-L1 cells as a model cell line. We hypothesized that the lignans would inhibit adipogenic differentiation in 3T3-L1 cells through the regulation of peroxisome proliferator-activated receptor γ (PPARγ). Our data indicate that sesamin and sesamolin inhibited the adipogenic differentiation of 3T3-L1 cells by dose-dependently decreasing lipid accumulation and triglyceride formation. Sesamin and sesamolin reduced the mRNA and protein expression of the adipogenesis-related transcription factors, PPARγ and CCAAT/enhancer-binding protein α, leading to the dose-dependent downregulations of their downstream targets, fatty acid binding protein 4, hormone-sensitive lipase, lipoprotein lipase, and glucose transporter 4. In addition, glucose uptake was dose-dependently attenuated by sesamin and sesamolin in both differentiated 3T3-L1 cells and HepG2 cells. Interestingly, our results suggested that sesamin and sesamolin might directly bind to PPARγ to inhibit its transcriptional activity. Finally, sesamin and sesamolin decreased the phosphorylation of 3 mitogen-activated protein kinase signaling components in differentiated 3T3-L1 cells. Taken together, our findings suggest that sesamin and sesamolin may exhibit antiobesity effects by potentially downregulating PPARγ and its downstream genes through the mitogen-activated protein kinase signaling pathway, offering important insights into the molecular mechanisms underlying the potential antiobesity effects of sesamin and sesamolin. The unresolved roles of sesamin/sesamolin on adipogenesis and antiobesity were studied using the 3T3-L1 preadipocytes to investigate the expression of key regulators for adipogenesis. A total of 20 to 80 µM sesamin/sesamolin effectively inhibited the activities of MAPK signaling pathways, downregulated PPARγ and C/EBPα and their downstream gene expression during preadipocyte differentiation. This in turn decreased lipid accumulation, lipid droplet formation, and glucose uptake. Abbreviations: C/EBPα, CCAAT/enhancer-binding protein α; FABP4, fatty acid binding protein 4; GLUT4, glucose transporter 4; HSL, hormone-sensitive lipase; LPL, lipoprotein lipase; MAPK, mitogen-activated protein kinase; MDI, 3-isobutyl-1-methylxanthine + dexamethasone + insulin; PPARγ, peroxisome proliferator-activated receptor γ. [Display omitted] [ABSTRACT FROM AUTHOR]

Tumor binding is an important parameter to derive unbound tumor concentration to explore pharmacokinetics (PK) and pharmacodynamics (PD) relationships for oncology disease targets. Tumor binding was evaluated using eleven matrices, including various commonly used ex vivo human and mouse xenograft and syngeneic tumors, tumor cell lines and liver as a surrogate tissue. The results showed that tumor binding is highly correlated among the different tumors and tumor cell lines except for the mouse melanoma (B16F10) tumor type. Liver fraction unbound (f u) has a good correlation with B16F10 tumor binding. Liver also demonstrates a two-fold equivalency, on average, with binding of other tumor types when a scaling factor is applied. Predictive models were developed for tumor binding, with correlations established with LogD (acids), predicted muscle f u (neutrals) and measured plasma protein binding (bases) to estimate tumor f u when experimental data are not available. Many approaches can be applied to obtain and estimate tumor binding values. One strategy proposed is to use a surrogate tumor tissue, such as mouse xenograft ovarian cancer (OVCAR3) tumor, as a surrogate for tumor binding (except for B16F10) to provide an early assessment of unbound tumor concentrations for development of PK/PD relationships. [ABSTRACT FROM AUTHOR]

Efficiently expanding Chinese hamster ovary (CHO) cells, which serve as the primary host cells for recombinant protein production, have gained increasing industrial significance. A significant hurdle in stable cell line development is the low efficiency of the target gene integrated into the host genome, implying the necessity for an effective screening and selection procedure to separate these stable cells. In this study, the genes of phenylalanine hydroxylase (PAH) and pterin 4 alpha carbinolamine dehydratase 1 (PCBD1), which are key enzymes in the tyrosine synthesis pathway, were utilized as selection markers and transduced into host cells together with the target genes. This research investigated the enrichment effect of this system and advanced further in understanding its benefits for cell line development and rCHO cell culture. A novel tyrosine-based selection system that only used PCBD1 as a selection marker was designed to promote the enrichment effect. Post 9 days of starvation, positive transductants in the cell pool approached 100%. Applied the novel tyrosine-based selection system, rCHO cells expressing E2 protein were generated and named CHO TS cells. It could continue to grow, and the yield of E2 achieved 95.95 mg/L in a tyrosine-free and chemically-defined (CD) medium. Herein, we introduced an alternative to antibiotic-based selections for the establishment of CHO cell lines and provided useful insights for the design and development of CD medium. [ABSTRACT FROM AUTHOR]

Despite the crucial role of peroxisomes in cellular redox maintenance, little is known about how these organelles transport redox metabolites across their membrane. In this study, we sought to assess potential associations between the cellular redox landscape and the human peroxisomal solute carrier SLC25A17, also known as PMP34. This carrier has been reported to function as a counter-exchanger of adenine-containing cofactors such as coenzyme A (CoA), dephospho-CoA, flavin adenine dinucleotide, nicotinamide adenine dinucleotide (NAD+), adenosine 3′,5′-diphosphate, flavin mononucleotide, and adenosine monophosphate. We found that inactivation of SLC25A17 resulted in a shift toward a more reductive state in the glutathione redox couple (GSSG/GSH) across HEK-293 cells, HeLa cells, and SV40-transformed mouse embryonic fibroblasts, with variable impact on the NADPH levels and the NAD+/NADH redox couple. This phenotype could be rescued by the expression of Candida boidinii Pmp47, a putative SLC25A17 orthologue reported to be essential for the metabolism of medium-chain fatty acids in yeast peroxisomes. In addition, we provide evidence that the alterations in the redox state are not caused by changes in peroxisomal antioxidant enzyme expression, catalase activity, H 2 O 2 membrane permeability, or mitochondrial fitness. Furthermore, treating control and Δ SLC25A17 cells with dehydroepiandrosterone, a commonly used glucose-6-phosphate dehydrogenase inhibitor affecting NADPH regeneration, revealed a kinetic disconnection between the peroxisomal and cytosolic glutathione pools. Additionally, these experiments underscored the impact of SLC25A17 loss on peroxisomal NADPH metabolism. The relevance of these findings is discussed in the context of the still ambiguous substrate specificity of SLC25A17 and the recent observation that the mammalian peroxisomal membrane is readily permeable to both GSH and GSSG. [Display omitted] • Loss of SLC25A17 selectively lowers the overall peroxisomal redox state. • Loss of SLC25A17 impacts peroxisomal NADPH metabolism. • The peroxisomal and cytosolic glutathione pools are kinetically disconnected. [ABSTRACT FROM AUTHOR]

• Tagetes minuta is a valuable medicinal plant with various uses, such as spice herb, traditional medicine, and a natural herbicide. • The study identified and quantified the secondary metabolites of T. minuta using colorimetric assays and liquid chromatography-mass spectrometry (LCMS). • The major classes of secondary metabolites identified in T. minuta were Phenolic acids, Flavonoids, Fatty acids, and Coumarins. • Ethanol extracts of T. minuta demonstrated higher antioxidant content, activity, and cytotoxicity against HepG2 and Vero cell lines, highlighting their pharmacological potential. The complexity of secondary metabolites in different medicinal plants, including Tagetes minuta , defines the general properties and uses of the plant. Tagetes minuta is reportedly a valuable medicinal plant that has long been used as a spice herb, medicine, a natural herbicide for weeds, and pest control in agriculture. In this study, the secondary metabolite profiles of the water and aqueous ethanol extracts of T. minuta were profiled through identification and quantification using colorimetric assays and a Waters Synapt G2 Quadrupole time-of-flight (QTOF) mass spectrometer (MS) connected to a Waters Acquity ultra-performance liquid chromatograph (UPLC). The antioxidant capacity of the extracts was evaluated with 2,2-Azino-di-3-ethylbenzthiazoline sulfonate and ferric reducing antioxidant power, as well as their toxicity on HepG2 (cancerous) and Vero (non-cancerous) cell lines. The major classes of secondary metabolites identified and quantified were phenolic acids (benzoic acids and hydroxycinnamic acids), flavonoids (chalcones, flavonols, flavanols, and flavones), fatty acids, coumarins and furanocoumarins. The bioavailability of these secondary metabolites is influenced by the polarity of the solvent of extraction. Extracts from aqueous ethanol showed higher secondary metabolites, corresponding to antioxidant content and activity as well as cytotoxicity in HepG2 and Vero cells, compared to the water extract. This study presents a comprehensive phytochemical knowledge of secondary metabolites, primarily phenolic compounds, identified in T. minuta, which can pave the way for future studies on the pharmacological importance and standardisation of the medicinal uses of the plant. [ABSTRACT FROM AUTHOR]

Cancer is a huge public health problem being one of the main causes of death globally. Specifically, melanoma is one of the most threatening cancer types due to the metastatic capacity, treatment resistance and mortality rates. It is evident the urgent need for research on new agents with pharmacological potential for cancer treatment, in order to develop new cancer therapeutic strategies and overcome drug resistance. The present work investigated the anti-tumoral potential of Chartergellus-CP1 peptide, isolated from Chartergellus communis wasp venom on human melanoma cell lines with different pigmentation degrees, namely the amelanotic cell line A375 and pigmented cell line MNT-1. Chartergellus-CP1 induced selective cytotoxicity to melanoma cell lines when compared to the lower induced cytotoxicity towards to nontumorigenic keratinocytes. Chartergellus-CP1 peptide induced apoptosis in both melanoma cell lines, cell cycle impairment in amelanotic A375 cells and intracellular ROS increase in pigmented MNT-1 cells. The amelanotic A375 cell line showed higher sensitivity to the peptide than the pigmented cell line MNT-1. From our knowledge, this is the first study reporting the cytotoxic effects of Chartergellus-CP1 on melanoma cells. [Display omitted] • Chartergellus-CP1 peptide induced selective anti-tumoral toxicity on melanoma cells. • Cytotoxic mechanisms are mainly related to oxidative stress and apoptotic death. • Chartergellus-CP1 may represent a novel therapeutic strategy on melanoma treatment. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the de facto standard host cells for biopharmaceuticals, and there is great interest in developing methods for constructing stable production cell lines. In this study, clones with a wide chromosome number distribution were selected from isolated antibody-producing strains, and subclones obtained from these clones were evaluated. The transgene copy number varied between the subclones. Even among subclones with similar copy numbers of antibody genes and maintained insertion sites, clones with different productivity were generated. Although the chromosome number distribution differed between these subclones, there was no correlation between the variability in chromosome number after cloning (genome instability) and productivity. Most of the subclones obtained from a parental strain with a wide chromosome number had the same wide chromosome number distribution as the parental strain. Less frequently, cells with less variation (remaining in one distribution) in chromosome number were isolated from cells with a wide chromosome number distribution, from which subclones with less variation in chromosome number were obtained when subcloning was performed again. These results imply that the characteristics of clones with chromosomal instability are inherited by subclones, and thus provide a better understanding of cell line stability/instability. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A (PP2A) is an essential tumor suppressor, with its activity often hindered in cancer cells by endogenous PP2A inhibitory proteins like SE translocation (SET). SET/PP2A axis plays a pivotal role in the colony-formation ability of cancer cells and the stabilization of c-Myc and E2F1 proteins implicated in this process. However, in osteosarcoma cell line HOS, SET knock-down (KD) suppresses the colony-formation ability without affecting c-Myc and E2F1. This study aimed to unravel the molecular mechanism through which SET enhances the colony-formation ability of HOS cells and determine if it is generalized to other cancer cells. Transcriptome analysis unveiled that SET KD suppressed mTORC1 signaling. SET KD inhibited Akt phosphorylation, an upstream kinase for mTORC1. PP2A inhibitor blocked SET KD-mediated decrease in phosphorylation of Akt and a mTORC1 substrate p70S6K. A constitutively active Akt restored decreased colonyformation ability by SET KD, indicating the SET/PP2A/Akt/mTORC1 axis. Additionally, enrichment analysis highlighted that Bmi-1, a polycomb group protein, is affected by SET KD. SET KD decreased Bmi-1 protein by Akt inhibition but not by mTORC1 inhibition, and exogenous Bmi-1 expression rescued the reduced colony formation by SET KD. Four out of eight cancer cell lines exhibited decreased Bmi-1 by SET KD. Further analysis of these cell lines revealed that Myc activity plays a role in SET KD-mediated Bmi-1 degradation. These findings provide new insights into the molecular mechanism of SET-regulated colony-formation ability, which involved Akt-mediated activation of mTORC1/p70S6K and Bmi-1 signaling. [ABSTRACT FROM AUTHOR]

• Hypobetalipoproteinemia disorders are characterized by lipid malabsorption. • There is no guidance about carotenoid supplementation in hypobetalipoproteinemias. • Carotenoid absorption is impaired in cells mimicking patient enterocytes. • Carotenoid plasma levels are dramatically low in patients compared to controls. • Evaluation of patient carotenoid status may help guide treatment. Familial hypobetalipoproteinemias (FHBL) are rare genetic diseases characterized by lipid malabsorption. We focused on abetalipoproteinemia (FHBL-SD1) and chylomicron retention disease (FHBL-SD3), caused by mutations in microsomal triglyceride transfer protein (MTTP) and SAR1B genes, respectively. Treatments include a low-fat diet and high-dose fat-soluble vitamin supplementations. However, patients are not supplemented in carotenoids, a group of lipid-soluble pigments essential for eye health. Our aim was to evaluate carotenoid absorption and status in the context of hypobetalipoproteinemia. We first used knock-out Caco-2/TC7 cell models of FHBL-SD1 and FHBL-SD3 to evaluate carotenoid absorption. We then characterized FHBL-SD1 and FHBL-SD3 patient status in the main dietary carotenoids and compared it to that of control subjects. In vitro results showed a significant decrease in basolateral secretion of α- and β-carotene, lutein, and zeaxanthin (-88.8 ± 2.2 % to -95.3 ± 5.8 %, -79.2 ± 4.4 % to -96.1 ± 2.6 %, -91.0 ± 4.5 % to -96.7 ± 0.3 % and -65.4 ± 3.6 % to -96.6 ± 1.9 %, respectively). Carotenoids plasma levels in patients confirmed significant deficiencies, with decreases ranging from –89 % for zeaxanthin to -98 % for α-carotene, compared to control subjects. Given the continuous loss in visual function despite fat-soluble vitamin treatment in some patients, carotenoid supplementation may be of clinical utility. Future studies should assess the correlation between carotenoid status and visual function in aging patients and investigate whether carotenoid supplementation could prevent their visual impairment. [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) are widely recognized as key drivers of cancer initiation, progression, and therapeutic resistance. Microfluidic chip technology offers a promising approach for CSC isolation and study. This study investigated the efficacy of a microfluidic chip-based method for isolating single cells from oral cancer cell lines characterized by high stem-like phenotypes. Specifically, the study focused on examining the sphere-forming capability and the expression of CSC markers, including aldehyde dehydrogenase 1A1 (ALDH1A1), CD44, and CD133, in isolated cell clones from OECM-1 and SAS cell lines. Oral cancer cell lines were subjected to isolation using a microfluidic chip. The captured single cells were cultured to assess their sphere-forming capacity in ultra-low binding culture. Furthermore, the protein expression levels of ALDH1A1, CD44, and CD133 in the isolated cell clones were analyzed using western blotting. The microfluidic chip-assisted isolation method significantly enhanced the sphere-forming capability of both OECM-1 and SAS cell clones compared to their parent cell lines. Moreover, the expression levels of CSC markers ALDH1A1, CD44, and CD133 were upregulated in the microfluidic chip-assisted isolated cell clones, indicating a higher stem-like phenotype. This study demonstrates the effectiveness of the microfluidic chip-based approach in isolating oral cancer cell clones with elevated stem-like characteristics. This method offers a valuable tool for further investigation of CSCs and their role in cancer progression, as well as future therapy development for oral cancers. [ABSTRACT FROM AUTHOR]

The natural compound betulin has been extensively explored for its potential application as an anticancer agent in recent years. Presently, the betulin compound was isolated from the brown seaweed, Sargassum ilicifolium. And further it was characterized through FT-IR, 13C NMR, 1H NMR, HPLC and ESI-MS analyses. Betulin alkane functional groups were identified by using FT-IR. Protons and their positions were confirmed by using 1H and 13C NMR spectra. Betulin has 15.59 % higher antioxidant contents with IC 50 value of >320 µg/mL. And also betulin has exhibited 76 %, 66.36 % (of viability) cytotoxicity respectively against the Hep-2 and THP-1 cancer cell lines. Amongst them, betulin has caused 67.25 % death in cervical cancer cell line with the highest cytotoxic percentage of 32.75 and IC 50 value of the betulin compound against the HeLa cell line was 31.32 µg/mL. Thus the betulin could form a potential source of anticancer agent. Presently, the betulin compound revealed high antioxidant capability with potential cytotoxic effect against cancer cell line. Present investigation showed the good antioxidant potential of the algal-betulin as well as the anticancer capacity especially against the HeLa cell line. Hence, further research is essential on this compound towards possible anticervical cancer-drug development. [Display omitted] • Betulin compound was purified from the brown seaweed, Sargassum ilicifolium. • It has revealed 15.59 % higher antioxidant contents. • Betulin has revealed a remarkable cytotoxicity of 76 % and 66.36 % against the Hep-2 and THP-1 cancer cell lines. • Betulin exhibited strong anti-cancer effects by causing 67.25 % of the cells in a cervical cancer cell line to die. [ABSTRACT FROM AUTHOR]

Recurrently observed aneuploidies across human cancers tend to be acquired early during tumorigenesis. Clustered regularly interspaced short palindromic repeats (CRISPR)-based tools allow targeted mis-segregation or deletion of individual chromosomes. Isogenic cellular models that differ in a single aneuploidy can reveal insights into underlying aneuploidy biology and their contributions to tumorigenesis. Multiple dosage-sensitive genes promote selection for aneuploid chromosomes in cancer. Chromosome copy number imbalances, otherwise known as aneuploidies, are a common but poorly understood feature of cancer. Here, we describe recent advances in both detecting and manipulating aneuploidies that have greatly advanced our ability to study their role in tumorigenesis. In particular, new clustered regularly interspaced short palindromic repeats (CRISPR)-based techniques have been developed that allow the creation of isogenic cell lines with specific chromosomal changes, thereby facilitating experiments in genetically controlled backgrounds to uncover the consequences of aneuploidy. These approaches provide increasing evidence that aneuploidy is a key driver of cancer development and enable the identification of multiple dosage-sensitive genes encoded on aneuploid chromosomes. Consequently, measuring aneuploidy may inform clinical prognosis, while treatment strategies that target aneuploidy could represent a novel method to counter malignant growth. [ABSTRACT FROM AUTHOR]

We describe the development and characterization of the (to date) smallest Natural Killer (NK) cell re-directing human B Cell Maturation Antigen (hBCMA) x CD16 dual engagers for potential treatment of multiple myeloma, based on combinations of small 58 amino acid, non-immunoglobulin, affibody affinity proteins. Affibody molecules to human CD16a were selected from a combinatorial library by phage display resulting in the identification of three unique binders with affinities (K D) for CD16a in the range of 100 nM–3 µM. The affibody exhibiting the highest affinity demonstrated insensitivity towards the CD16a allotype (158F/V) and did not interfere with IgG (Fc) binding to CD16a. For the construction of hBCMA x CD16 dual engagers, different CD16a binding arms, including bi-paratopic affibody combinations, were genetically fused to a high-affinity hBCMA-specific affibody. Such 15–23 kDa dual engager constructs showed simultaneous hBCMA and CD16a binding ability and could efficiently activate resting primary NK cells and trigger specific lysis of a panel of hBCMA-positive multiple myeloma cell lines. Hence, we report a novel class of uniquely small NK cell engagers with specific binding properties and potent functional profiles. [Display omitted] • A novel and uniquely small class of BCMA x CD16a dual engagers is described. • Based on combinations of affibody affinity proteins developed by combinatorial protein engineering. • Bi-paratopic binding to CD16a results in higher affinity. • Dual engagers are effective in activating resting NK cells and inducing a BCMA-dependent killing of MM cells. [ABSTRACT FROM AUTHOR]

• MO and TV caused a decrease in NF-κβ immunoreactivity in the SW480 colon cancer cell line. • It showed that MO and TV have effects on inflammation and signaling pathways that control cell cycle and apoptosis. • It also induced damage in cancer cells with an increased number of TUNEL positive cells and higher TNF-α and IL-6 immunoreactivity. The creation of novel therapeutic approaches and more potent chemotherapeutic agents is the current emphasis of research into cancer therapy. About 60% of drugs used for cancer treatment are isolated from natural products, and the plant kingdom has been the most important resource. The study aimed to evaluate the cytotoxicity of Melissa officinalis L. (MO) and Thymus vulgaris L. (TV) extracts against SW480 cell lines, to elucidate cell death mechanisms and to determine their effect on inflammation. The effect of MO and TV on cell viability was evaluated using an MTT assay in SW480 cell line. After MO and TV treatment, apoptosis was evaluated by Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. An indirect peroxidase technique for immunocytochemical analysis of related proteins such as IL-6, TNF-α, and NF-kB was done. Significant differences in cell viability were observed 24 hours after MO and TV application in the cell line. In terms of TUNEL+ apoptotic cells, there was a statistically significant difference between the control group and the IC50 group in SW480 cell lines treated with both MO and TV. IL-6 and TNF-α, expression levels were found to be significantly lower after MO and TV administration in a concentration-dependent manner, whereas NF-kB expression was upregulated. Our findings show that it is effective on inflammation and signaling pathways that control the cell cycle and apoptosis, but more studies are needed to determine what mechanistic effect the active components of these plants have. [ABSTRACT FROM AUTHOR]

A therapeutic system with the ability to target more than one protein is an important aim of cancer therapy since tumor growth is accompanied by dysregulation of many biological pathways. G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich DNA or RNA oligonucleotides, with the ability to bind to different targets. In this study, we constructed ten novel bispecific G-quadruplex conjugates based on AT11, TBA, T40214 and T40231 aptamer structures, with the ability to bind two different targets at once in cancer cells. We analyzed the physicochemical aspects and the anticancer properties of novel molecules relating them with the single G-quadruplex unit and attempted to comprehend the correlation between the structures of bispecific G-quadruplexes with their biological activity. Our studies uncovered conjugates with considerable antiproliferative potential in HeLa and MCF-7 cancer cell lines, however with relatively low thermal stability or low nuclease resistance. Three conjugates among all studied oligonucleotides possess improved antiproliferative activity in MCF-7 cell line in comparison to their single G-quadruplex units leading to up to 90% inhibition of cancer cells growth, but their inhibitory potential is rather comparable to the effect observed for mix of two separate G-quadruplex units. Importantly, the conjugation enhances oligonucleotides enzymatic stability leading to the improvement of their therapeutic profile. The comprehensive studies presented herein indicate new approach for possibly effective cancer therapy and for the design of G4-based drugs. • Novel bispecific G-quadruplex conjugates containing two domains are developed. • G-quadruplexes have the ability to bind to two different targets. • Bispecific G-quadruplexes were characterized physicochemically and biologically. • Novel molecules have considerable anticancer activity in HeLa and MCF-7 cell lines. • The conjugation enhances oligonucleotides enzymatic stability. [ABSTRACT FROM AUTHOR]

New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Breast cancer is associated with high morbidity and mortality in women since conventional treatment methods are limited due to toxicity problems and inadequate targeting of cancer cells. The aim of this study is to develop caffeic acid phenethyl ester (CAPE) loaded polymeric nanoparticles to provide treatment for breast cancer and investigate their anti-tumor activity on MCF-7 and MDA-MB-231 breast cancer cells. CAPE was encapsulated into poly lactic-co-glycolic acid (PLGA) polymer using single-emulsion (o/w) solvent evaporation method and characterization of CAPE loaded PLGA nanoparticles (CAPE-NPs) were performed by particle size, zeta potential, polydispersity index , in vitro release profile, and surface morphology. CAPE-NPs were tested for cytotoxicity, anti-migration, anti-proliferation and apoptotic activities on breast cancer cells. The mean nanoparticle size was found as 196,1 ± 8982 nm and polydispersity index (PDI) was 0186 ± 0069. The results of the in vitro cell culture studies indicated that treatment of breast cancer cells with CAPE and CAPE-NPs inhibited cell viability and proliferation. IC 50 values for 48 h were determined as 28.35 μg/ml,19.57 μg/ml for CAPE and CAPE-NP, respectively. Additionally, CAPE-NPs induced intrinsic pathway of apoptosis in MCF-7 and MDA-MB-231 cell lines. Overall, CAPE-NPs showed more efficient anti-tumour activity on breast cancer cells by small size and enhanced bioavability. [Display omitted] • The aim of this study is to develop CAPE-NPs to provide treatment for breast cancer. • CAPE loaded PLGA nanoparticles were produced (CAPE-NPs). • CAPE-NPs anti-tumour activity was evaluated on breast cancer cell lines. • CAPE-NPs exhibited higher cytotoxicity than CAPE due to their small size and controlled release. • CAPE-NPs demonstrated a decrease in cell proliferation and induced the intrinsic pathway of apoptosis. [ABSTRACT FROM AUTHOR]

To date, long-term rodent carcinogenesis assays are the only assays recognized by regulators to assess non-genotoxic carcinogens, but their reliability has been questioned. In vitro cell transformation assays (CTAs) could represent an interesting alternative to animal models as it has the advantage of detecting both genotoxic and non-genotoxic transforming chemicals. Among them, Bhas 42 CTA uses a cell line that has been transfected with the oncogenic sequence v-Ha- ras. This sequence confers an "initiated" status to these cells and makes them particularly sensitive to non-genotoxic agents. In a previous work, transcriptomic analysis revealed that the treatment of Bhas 42 cells with transforming silica (nano)particles and 12- O -tetradecanoylphorbol-13-acetate (TPA) commonly modified the expression of 12 genes involved in cell proliferation and adhesion. In the present study, we assess whether this signature would be the same for four other soluble transforming agents, i.e. mezerein, methylarsonic acid, cholic acid and quercetin. The treatment of Bhas 42 cells for 48 h with mezerein modified the expression of the 12 genes of the signature according to the same profile as that of the TPA. However, methylarsonic acid and cholic acid gave an incomplete signature with changes in the expression of only 7 and 5 genes, respectively. Finally, quercetin treatment induced no change in the expression of all genes but exhibited higher cytotoxicty. These results suggest that among the transforming agents tested, some may share similar mechanisms of action leading to cell transformation while others may activate different additional pathways involved in such cellular process. More transforming and non-transforming agents and gene markers should be tested in order to try to identify a relevant gene signature to predict the transforming potential of non-genotoxic agents. • A 12-gene signature predictive of cell transformation in Bhas 42 cells was investigated. • TPA, mezerein, methylarsonic acid, cholic acid and quercetin were tested. • The signature could be reduced to Prl2c1, Postn , Timp4 and Lum genes. • Dose-response in gene expression changes were not always found. • The relevance of theses genes as predictive biomarkers needs to be confirmed. [ABSTRACT FROM AUTHOR]

The role of metformin on the radiosensitising effect of cisplatin is not clear. Here we investigated the radiosensitising effect of metformin alone and combined with cisplatin in HeLa cells, as well as the implications of the adenosine monophosphate-activated protein kinase (AMPK) pathway on the radiosensitising effect. HeLa cells were treated with ionising radiation, metformin, cisplatin, A769662 (AMPK activator) and dorsomorphin (AMPK inhibitor) or in combination. A cell proliferation assay, Western blot and flow cytometry were carried out. Metformin potentiated cisplatin cytotoxicity when administered 4 h before ionising radiation. Although the radiosensitising effects of metformin and cisplatin alone were observed, which is more apparent at high ionising radiation doses, the metformin–cisplatin combination did not increase the radiosensitivity of cisplatin at any ionising radiation dose. Dorsomorphin alone significantly decreased cell proliferation and potentiated the radiosensitising effects of cisplatin with ionising radiation. Administration of A769662 24 h prior to cisplatin treatment resulted in an increased AMPK level that yielded resistance to cisplatin, but this effect was not observed in HeLa cells concomitantly treated with A769662 and cisplatin. Modulation of AMPK may have a role in cervical cancer treatment. Increased AMPK levels result in higher sensitivity to ionising radiation but causes resistance to cisplatin. Dorsomorphin is proven to be a potent radiosensitising agent. The use of metformin alone may be an option as a radiosensitiser during high-dose ionising radiation (e.g. intracavitary brachytherapy). • Increased AMPK levels result in higher sensitivity to ionising radiation, while causing resistance to cisplatin. • Dorsomorphin proves to be a potent radiosensitising agent. • Metformin alone may be an option as a radiosensitiser during high-dose ionising radiation (e.g. intracavitary brachytherapy). [ABSTRACT FROM AUTHOR]

The development of safe and effective second-generation COVID-19 vaccines to improve affordability and storage stability requirements remains a high priority to expand global coverage. In this report, we describe formulation development and comparability studies with a self-assembled SARS-CoV-2 spike ferritin nanoparticle vaccine antigen (called DCFHP), when produced in two different cell lines and formulated with an aluminum-salt adjuvant (Alhydrogel, AH). Varying levels of phosphate buffer altered the extent and strength of antigen-adjuvant interactions, and these formulations were evaluated for their (1) in vivo performance in mice and (2) in vitro stability profiles. Unadjuvanted DCFHP produced minimal immune responses while AH-adjuvanted formulations elicited greatly enhanced pseudovirus neutralization titers independent of ∼100%, ∼40% or ∼10% of the DCFHP antigen adsorbed to AH. These formulations differed, however, in their in vitro stability properties as determined by biophysical studies and a competitive ELISA for measuring ACE2 receptor binding of AH-bound antigen. Interestingly, after one month of 4°C storage, small increases in antigenicity with concomitant decreases in the ability to desorb the antigen from the AH were observed. Finally, we performed a comparability assessment of DCFHP antigen produced in Expi293 and CHO cells, which displayed expected differences in their N-linked oligosaccharide profiles. Despite consisting of different DCFHP glycoforms, these two preparations were highly similar in their key quality attributes including molecular size, structural integrity, conformational stability, binding to ACE2 receptor and mouse immunogenicity profiles. Taken together, these studies support future preclinical and clinical development of an AH-adjuvanted DCFHP vaccine candidate produced in CHO cells. [ABSTRACT FROM AUTHOR]

Including active renal excretion in physiologically based kinetic (PBK) models can improve their use in quantitative in vitro- in vivo extrapolation (QIVIVE) as a new approach methodology (NAM) for predicting the acute toxicity of organic cation transporter 2 (OCT2) substrates like paraquat (PQ). To realise this NAM, kinetic parameters Vmax and Km for in vitro OCT2 transport of PQ were obtained from the literature. Appropriate scaling factors were applied to translate the in vitro Vmax to an in vivo Vmax. in vitro cytotoxicity data were defined in the rat RLE-6TN and L2 cell lines and the human A549 cell line. The developed PQ PBK model was used to apply reverse dosimetry for QIVIVE translating the in vitro cytotoxicity concentration-response curves to predicted in vivo toxicity dose-response curves after which the lower and upper bound benchmark dose (BMD) for 50% lethality (BMDL 50 and BMDU 50) were derived by applying BMD analysis. Comparing the predictions to the in vivo reported LD 50 values resulted in a conservative prediction for rat and a comparable prediction for human showing proof of principle on the inclusion of active renal excretion and prediction of PQ acute toxicity for the developed NAM. [Display omitted] • A new approach methodology (NAM) predicting acute paraquat toxicity was defined. • This NAM included a newly developed PBK model with active renal excretion via OCT2. • Cytotoxicity data in lung epithelial cell lines were used for QIVIVE. • QIVIVE was based on PBK model based reverse dosimetry. • The in vivo dose response curves adequately predicted acute paraquat toxicity. [ABSTRACT FROM AUTHOR]