In a previous work, we proposed a vaccine chimeric antigen based on the fusion of the SARS-CoV-2 N protein to the extracellular domain of the human CD40 ligand (CD154). This vaccine antigen was named N-CD protein and its expression was carried out in HEK-293 stably transfected cells, grown in adherent conditions and serum-supplemented medium. The chimeric protein obtained in these conditions presented a consistent pattern of degradation. The immunization of mice and monkeys with this chimeric protein was able to induce a high N-specific IgG response with only two doses in pre-clinical experiments. In order to explore ways to diminish protein degradation, in the present work, the N and N-CD proteins were produced in suspension cultures and serum-free media following transient transfection of the HEK-293 clone 3F6, at different scales, including stirred-tank controlled bioreactors. The results showed negligible or no degradation of the target proteins. Further, clones stably expressing N-CD were obtained and adapted to suspension culture, obtaining similar results to those observed in the transient expression experiments in HEK-293-3F6. The evidence supports transient protein expression in suspension cultures and serum-free media as a powerful tool to produce in a short period of time high levels of complex proteins susceptible to degradation, such as the SARS-CoV-2 N protein.

BACKGROUND: Apoptotic agents from natural products like phenolic compounds can be used effectively in the treatment of cancer. Chlorogenic acid (CGA) is one of the phenolic compounds in medicinal plants with anti-cancer properties. In this research, we aimed to explore the anti-cancer mode of action of CGA on colorectal cancer (CRC) cells in vitro conditions.
METHODS: HT-29 and HEK-293 cells were cultured after MTT assay for 24 h with CGA 100 µM, and without CGA. Then, flow cytometry assays and the expression of apoptosis-related genes including caspase 3 and 9, Bcl-2 and Bax, and cell cycle-related genes including P21, P53 and NF-κB at mRNA and protein levels were examined. Finally, we measured the amount of intracellular reactive oxygen species (ROS).
RESULTS: The cell viability of all two-cell lines decreased in a dose-dependent manner. Moreover, CGA induces cell cycle arrest in HT-29 cells by increasing the expression of P21 and P53. It also induces apoptosis in HT-29 cells by mitigating Bcl-2 and NF-κB expression and elevating caspase 3 and 9 expression and ROS levels.
CONCLUSIONS: Considering the cytotoxicity and cell cycle arrest and induction of apoptosis in the colon cancer cell line by CGA, it can be concluded that CGA is a suitable option for the treatment of colon cancer.

Parkinson\'s disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-β-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.

BACKGROUND: Sharbat-e-bazoori Motadil (SBM) is a polyherbal formulation that have been used for centuries as a part of the Unani system of medicine for renal disease.
OBJECTIVE: The objective of this study was to explore and validate the nephroprotective potential of sugar-free SBM (SF-SBM) and its mechanisms of action against sodium fluoride (NaF)-induced nephrotoxicity in HEK-293 cells. Additionally, the study aimed to assess the quality control of SF-SBM and investigate its effects using an in vivo rat model with pattern recognition following oral administration of SF-SBM.
METHODS: The nephroprotective effect of SF-SBM was investigated using both an HEK-293 cell line and Wistar rats. Nephrotoxicity was induced in these models by administering NaF at a concentration of 600 ppm (parts per million) for a duration of seven days. The SF-SBM formulation was standardized using high-performance thin-layer chromatography (HPTLC) to assess the presence of marker compounds, namely gallic acid, quercetin, and ferulic acid. Metabolite characterization of SF-SBM was carried out using ultra-high-performance liquid chromatography mass spectrometry (UPLC-MS) with a monolithic capillary silica-based C18 column. This analytical technique allowed for the identification of bioactive substances and verification of the identified markers. Acute toxicity of SF-SBM was evaluated in Wistar rats by administering a single oral dose of 2000 mg/kg of SF-SBM. The nephroprotective efficacy of SF-SBM was further assessed at low (LD), medium (MD) and high (HD) doses of 32.1, 64.2, and 128.4 mg/kg, respectively, administered orally. Nephrotoxicity was induced in Wistar rats by adding NaF to their drinking water for seven days. Biochemical and urine markers were analyzed to evaluate the antioxidant, inflammatory, and apoptotic potential of SF-SBM. Additionally, histopathological analysis and immunohistochemical alterations in the expression of caspase-3 and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-4 (NOX-4) in kidney tissue were performed to confirm the findings of the in vivo experiments. Furthermore, in vivo pattern recognition of SF-SBM metabolites, identified through GC-MS metabolomics, and in-silico docking analysis of major metabolites in plasma were conducted to gain further insights.
RESULTS: Phytochemical analysis using HPTLC, TLC-bioautography, and UPLC-MS revealed the presence of several bioactive constituents in SF-SBM, including ferulic acid, gallic acid (GA), ellagic acid, quercetin, and apigenin. These compounds exhibit diverse pharmacological properties. In vitro studies demonstrated the protective effect of SF-SBM on HEK-293 cell line against nephrotoxicity. The acute toxicity study of SF-SBM at a dose of 2000 mg/kg showed no mortality or signs of toxicity throughout the 14-day observation period. In the in vivo studies, administration of NaF resulted in significant elevation (P < 0.001) of biochemical and urine parameters, indicating oxidative, inflammatory, and apoptotic stress. Histopathological examination revealed severe depletion of Bowman\'s capsule, and immunohistochemistry demonstrated negative immunostaining for caspase-3 and reduced NOX-4 reactions. Pre-treatment with SF-SBM significantly attenuated the elevated biochemical and urine markers, restored the antioxidant enzyme levels (such as SOD, CAT, GSH, GPx and NO), and regulated the expression of inflammatory cytokines (TNF-α, IL-1β, CASP-3) in kidney tissue at doses of SF-SBM-MD (64.2 mg/kg) and SF-SBM-HD (128.4 mg/kg), showing comparable results to those of α-Ketoanalogue. Histopathological assessment demonstrated improvements in tissue damage. Pattern recognition analysis of SF-SBM identified the presence of 56 metabolites at different time intervals. Additionally, in-silico studies revealed strong interactions of SF-SBM with a binding energy of -6.5 and -5.6 kcal for 4C2N.
CONCLUSIONS: The phytoconstituents present in SF-SBM play a crucial role in its nephroprotective action by acting as potent antioxidants and reducing proinflammatory and apoptotic damage in rat cells. This indicates that SF-SBM has promising potential for the treatment of nephrotoxicity.

Titanium dioxide nanoparticles (TiO2 NPs) have been widely used in food, cosmetics, and biomedical research. However, human safety following exposure to TiO2 NPs remains to be fully understood. The aim of this study was to evaluate the in vitro safety and toxicity of TiO2 NPs synthesized via the Stöber method under different washing and temperature conditions. TiO2 NPs were characterized by their size, shape, surface charge, surface area, crystalline pattern, and band gap. Biological studies were conducted on phagocytic (RAW 264.7) and non-phagocytic (HEK-239) cells. Results showed that washing amorphous as-prepared TiO2 NPs (T1) with ethanol while applying heat at 550 °C (T2) resulted in a reduction in the surface area and charge compared to washing with water (T3) or a higher temperature (800 °C) (T4) and influenced the formation of crystalline structures with the anatase phase in T2 and T3 and rutile/anatase mixture in T4. Biological and toxicological responses varied among TiO2 NPs. T1 was associated with significant cellular internalization and toxicity in both cell types compared to other TiO2 NPs. Furthermore, the formation of the crystalline structure induced toxicity independent of other physicochemical properties. Compared with anatase, the rutile phase (T4) reduced cellular internalization and toxicity. However, comparable levels of reactive oxygen species were generated following exposure to the different types of TiO2, indicating that toxicity is partially driven via non-oxidative pathways. TiO2 NPs were able to trigger an inflammatory response, with varying trends among the two tested cell types. Together, the findings emphasize the importance of standardizing engineered nanomaterial synthesis conditions and evaluating the associated biological and toxicological consequences arising from changes in synthesis conditions.

Over the past decades, virus-like particle (VLP)-based gene therapy (GT) evolved as a promising approach to cure inherited diseases or cancer. Tremendous costs due to inefficient production processes remain one of the key challenges despite considerable efforts to improve titers. This review aims to link genome-scale metabolic models (GSMMs) to cell lines used for VLP synthesis for the first time. We summarize recent advances and challenges of GSMMs for Chinese hamster ovary (CHO) cells and provide an overview of potential cell lines used in GT. Although GSMMs in CHO cells led to significant improvements in growth rates and recombinant protein (RP)-production, no GSMM has been established for VLP production so far. To facilitate the generation of GSMM for these cell lines we further provide an overview of existing omics data and the highest production titers so far reported.

Despite the significant advancements in complex anticancer therapy, the search for new and more efficient specific anticancer agents remains a top priority in the field of drug discovery and development. Here, based on the structure-activity relationships (SARs) of eleven salicylaldehyde hydrazones with anticancer activities, we designed three novel derivatives. The compounds were tested in silico for drug-likeness, synthesized, and evaluated in vitro for anticancer activity and selectivity on four leukemic cell lines (HL-60, KE-37, K-562, and BV-173), one osteosarcomic cell line (SaOS-2), two breast adenocarcinomic cell lines (MCF-7 and MDA-MB-231), and one healthy cell line (HEK-293). The designed compounds were found to have appropriate drug likeness and showed anticancer activities in all cell lines tested; particularly, two of them exhibited remarkable anticancer activity in nanomolar concentrations on the leukemic cell lines HL-60 and K-562 and the breast cancer MCF-7 cells and extraordinary selectivity for the same cancer lines ranging between 164- and 1254-fold. The study also examined the effects of different substituents on the hydrazone scaffold and found that the 4-methoxy salicylic moiety, phenyl, and pyridinyl rings are the most appropriate for anticancer activity and selectivity of this chemical class.

Bioelectrical variations trigger different cell responses, including migration, mitosis, and mutation. At the tissue level, these actions result in phenomena such as wound healing, proliferation, and pathogenesis. Monitoring these mechanisms dynamically is highly desirable in diagnostics and drug testing. However, existing technologies are invasive: either they require physical access to the intracellular compartments, or they imply direct contact with the cellular medium. Here, we present a novel approach for the passive recording of electrical signals from non-excitable cells adhering to 3D microelectrodes, based on optical mirroring. Preliminary results yielded a fluorescence intensity output increase of the 5,8% in the presence of a HEK-293 cell on the electrode compared to bare microelectrodes. At present, this technology may be employed to evaluate cell-substrate adhesion and monitor cell proliferation. Further refinements could allow extrapolating quantitative data on surface charges and resting potential to investigate the electrical phenomena involved in cell migration and cancer progression.

UNASSIGNED: As an environmental contaminant, Arsenic (As) poses many risks to human health. Increased Oxidative Stress (OS) and decreased antioxidant cell defense are the suggested mechanisms of carcinogenicity and toxicity of As. As a powerful antioxidant and water-soluble compound, vitamin C protects cells and tissues against oxidation and has a wide range of healing properties.
UNASSIGNED: The current study aimed to formulate a suitable ascorbic acid (vitamin C) niosome and compare it with vitamin C in preventing As-induced toxicity in HEK-293 cells.
UNASSIGNED: Various formulas of vitamin C niosomes were prepared by C-SPAN mixed with cholesterol. The physicochemical characteristics of niosomal formulations, including load size, zeta-potential, and the drug release profile, were evaluated in HEK-293 cells. Then, OS biomarkers such as total reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), Antioxidant Capacity (TAC), and superoxide dismutase (SOD) activities determined the protective effects of vitamin C niosomes compared with vitamin C against As-induced toxicity.
UNASSIGNED: The particle size and zeta potential of the optimal vitamin C niosome were 163.2 ± 6.1 nm and 23.3 ± 3.5 mV, respectively. Arsenic increased ROS and MDA levels while decreasing CAT, TAC, and SOD activities in the HEK-293 cell line. Finally, the vitamin C niosome decreased OS and increased antioxidant properties more than vitamin C.
UNASSIGNED: Vitamin C niosome was more effective than vitamin C in treating As-induced toxicity in vitro.

Human stem cell factor (hSCF) is an early-acting growth factor that promotes proliferation, differentiation, migration, and survival in several tissues. It plays a crucial role in hematopoiesis, gametogenesis, melanogenesis, intestinal motility, and in development and recovery of nervous and cardiovascular systems. Potential therapeutic applications comprise anemia treatment, mobilization of hematopoietic stem/progenitor cells to peripheral blood, and increasing gene transduction efficiency for gene therapy. Developing new tools to characterize recombinant hSCF in most native-like form as possible is crucial to understand the complexity of its in vivo functions and for improving its biotechnological applications. The soluble domain of hSCF was expressed in HEK293 cells. Highly purified rhSCF showed great molecular mass variability due to the presence of N- and O-linked carbohydrates, and it presented a 2.5-fold increase on proliferative activity compared to bacteria-derived hSCF. Three hybridoma clones producing monoclonal antibodies (mAbs) with high specificity for the glycoprotein were obtained. 1C4 and 2D3 mAbs were able to detect bacteria-derived and glycosylated rhSCF and demonstrated to be excellent candidates to develop a sandwich ELISA assay for rhSCF quantification, with detection limits of 0.18 and 0.07 ng/ml, respectively. Interestingly, 1A10 mAb only recognized glycosylated rhSCF, suggesting that sugar moieties might be involved in epitope recognition. 1A10 mAb showed the highest binding affinity, and it constituted the best candidate for immunodetection of the entire set rhSCF glycoforms in western blot assays, and for intracellular cytokine staining. Our work shows that combining glycosylated rhSCF expression with hybridoma technology is a powerful strategy to obtain specific suitable immunochemical assays and thus improve glycoprotein-producing bioprocesses. KEY POINTS: • Soluble glycosylated human SCF exerted improved proliferative activity on UT-7 cells. • Three mAbs with high specificity targeting glycosylated human SCF were obtained. • mAbs applications comprise sandwich ELISA, western blot, and immunofluorescence assays.